U.S. patent number 5,834,229 [Application Number 08/330,161] was granted by the patent office on 1998-11-10 for nucleic acids vectors and host cells encoding and expressing heregulin 2-.alpha..
This patent grant is currently assigned to Genentech, Inc.. Invention is credited to William E. Holmes, Richard L. Vandlen.
United States Patent |
5,834,229 |
Vandlen , et al. |
November 10, 1998 |
Nucleic acids vectors and host cells encoding and expressing
heregulin 2-.alpha.
Abstract
Novel 2 polypeptides with binding affinity for the p185.sup.HER2
receptor, designated heregulin 2-.alpha. and heregulin 2-.beta.,
have been identified and purified from human tissue. The cDNA
encoding the novel heregulin 2-.alpha. has been isolated from human
tissue and sequenced. Provided herein is nucleic acid sequence of
the heregulin 2-.alpha. useful in the production of heregulin
2-.alpha. by recombinant means. Further provided an amino acid
sequence of heregulin 2-.alpha. and heregulin 2-.beta.. Heregulins
and their antibodies are useful as therapeutic agents and in
diagnostic methods.
Inventors: |
Vandlen; Richard L.
(Hillsborough, CA), Holmes; William E. (Pacifica, CA) |
Assignee: |
Genentech, Inc. (South San
Francisco, CA)
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Family
ID: |
26712121 |
Appl.
No.: |
08/330,161 |
Filed: |
October 25, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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35430 |
Mar 22, 1993 |
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705256 |
May 24, 1991 |
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Current U.S.
Class: |
435/69.1;
435/91.2; 435/325; 435/252.3; 536/23.5; 536/24.33; 536/24.31;
435/320.1 |
Current CPC
Class: |
C07K
14/4756 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
14/475 (20060101); C07K 14/435 (20060101); A61K
38/00 (20060101); C12N 015/00 (); C12N 015/85 ();
C12N 007/00 (); C12N 015/63 (); C07H 021/04 () |
Field of
Search: |
;435/91.2,969.1,320.1,240.2,252.3,252.8 ;536/23.5,24.33,24.31 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0505148 |
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Sep 1993 |
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EP |
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92/00595 |
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Apr 1992 |
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GB |
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WO91/15230 |
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Oct 1971 |
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WO |
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WO 92/18627 |
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Oct 1992 |
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WO |
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9322339 |
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Nov 1993 |
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WO |
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WO 93/22424 |
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Nov 1993 |
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WO |
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Other References
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|
Primary Examiner: Arthur; Lisa B.
Attorney, Agent or Firm: Lee; Wendy M.
Parent Case Text
CROSS REFERENCES
This application is a continuation of U.S. patent application Ser.
No. 08/035,430 filed 22, Mar. 1993, now abandoned, which
application is a continuation of U.S. patent application Ser. No.
07/705,256 filed 24 May 1991, now abandoned, which applications are
incorporated herein by reference and to which applications priority
is claimed under 35 USC .sctn.120.
Claims
We claim:
1. An isolated polynucleotide encoding a polypeptide comprising the
amino acid sequence
-Ala-Glu-Lys-Glu-Lys-Thr-Phe-Cys-Val-Asn-Gly-Gly-Glu-Cys-Phe-Met-Val-Lys-A
sp-Leu-Ser-Asn-Pro-, wherein the polypeptide is able to stimulate
tyrosine phosphorylation of the p185.sup.HER2 receptor in
MDA-MB-453 cells.
2. The polynucleotide of claim 1 wherein the polypeptide comprises
an amino acid sequence extending from about from S216 to A227, to
about from K272 to R286 within SEQ ID NO:11.
3. The polynucleotide of claim 1 wherein the polypeptide comprises
an amino acid sequence extending from C226 to C265 within SEQ ID
NO:11.
4. The polynucleotide of claim 1 wherein the polypeptide comprises
an amino acid sequence extending from an arginine, lysine, valine
or methionine or adjacent residue within A1 to C226, to an
arginine, lysine, valine or methionine or adjacent residue within
C265 to K285 within SEQ ID NO:11.
5. The polynucleotide of claim 4 wherein the amino acid sequence
comprises a sequence extending to K272, K278, or K285.
6. The polynucleotide of claim 1 which further encodes an
immunogenic polypeptide which elicits antibodies in an animal
immunized therewith.
7. The polynucleotide of claim 1 which further encodes an
immunoglobulin constant region, albumin or ferritin.
8. A recombinant expression vector comprising the polynucleotide of
claim 1.
9. A method for making a polypeptide comprising culturing a host
cell transfected to express the polynucleotide of claim 1 and
recovering the polypeptide from the host cell.
10. The method of claim 9 wherein the polypeptide is recovered from
the host cell culture medium.
11. The method of claim 9 wherein the host cell is transfected with
a recombinant expression vector comprising the polynucleotide.
12. A method of amplifying the polynucleotide of claim 1 comprising
performing a polymerase chain reaction such that the polynucleotide
is amplified.
13. An isolated polynucleotide encoding a polypeptide comprising
the amino acid sequence of mature heregulin 2-alpha within SEQ ID
NO:11.
14. A recombinant expression vector comprising the polynucleotide
of claim 13.
15. A host cell transformed with the vector of claim 14.
16. A method for making a polypeptide comprising:
(a) culturing a host cell comprising the polynucleotide of claim 13
such that the polynucleotide is expressed; and
(b) recovering the polypeptide from the cell culture.
17. An isolated polynucleotide encoding a polypeptide comprising
the amino acid sequence of heregulin 2-alpha of SEQ ID NO:11
lacking a functional transmembrane domain, wherein the polypeptide
is able to stimulate tyrosine phosphorylation of the p185.sup.HER2
receptor in MDA-MB-453 cells.
18. A polynucleotide encoding a polypeptide comprising the amino
acid sequence of mature heregulin 2-alpha within SEQ ID NO:11 under
the control of an exogenous promoter which is heterologous to
heregulin 2-alpha.
19. An isolated polynucleotide which specifically hybridizes under
stringent conditions with the complement of the polynucleotide of
SEQ ID NO:10 and encodes a polypeptide which is able to stimulate
tyrosine phosphorylation of the p185.sup.HER2 receptor in
MDA-MB-453 cells.
20. An isolated polynucleotide which specifically hybridizes under
stringent conditions with the complement of a polynucleotide
encoding the growth factor domain (GFD) of heregulin 2-alpha within
SEQ ID NO:11, wherein the isolated polynucleotide encodes a
polypeptide which able to stimulate tyrosine phosphorylation of the
p185.sup.HER2 receptor in MDA-MB-453 cells.
21. The polynucleotide of claim 20 which encodes human heregulin
2-alpha.
22. The polynucleotide of claim 20 wherein stringent conditions
comprise washing with 0.015M NaCl/0.0015M sodium citrate/0.1%
NaDodSO4 at 50 degrees C.
23. The polynucleotide of claim 20 wherein stringent conditions
comprise hybridizing at 42 degrees C. in the presence of 50%
vol/vol formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, 50 mM sodium phosphate, 750 mM NaCl, 75 mM
sodium citrate, pH 6.5 buffer.
24. The polynucleotide of claim 20 wherein stringent conditions
comprise hybridizing at 42 degrees C. in the presence of 50%
formamide, 5.times.SSC (0.75M NaCl, 0.075M sodium citrate), 50 mM
sodium phosphate, 5.times.Denhardt's solution, sonicated salmon
sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate, with washes
at 42 degrees C. in 0.2.times.SSC and 0.1% SDS.
25. A recombinant expression vector comprising the polynucleotide
of claim 20.
26. A host cell comprising the polynucleotide of claim 20.
27. A method for making a polypeptide comprising:
(a) culturing a host cell comprising the polynucleotide of claim 20
such that the polynucleotide is expressed; and
(b) recovering the polypeptide from the cell culture.
28. An isolated polynucleotide which specifically hybridizes under
stringent conditions with the complement of a polynucleotide
encoding heregulin 2-alpha of SEQ ID NO:11, wherein the
polynucleotide encodes a polypeptide which is able to stimulate
tyrosine phosphorylation of the p185.sup.HER2 receptor in
MDA-MB-453 cells.
29. An isolated polynucleotide encoding a polypeptide comprising an
amino acid sequence extending from C226 to C240 within SEQ ID
NO:11, wherein the polypeptide is able to stimulate tyrosine
phosphorylation of the p185.sup.HER2 receptor in MDA-MB-453
cells.
30. An isolated polynucleotide encoding a polypeptide comprising an
amino acid sequence extending from C234 to C254 within SEQ ID
NO:11, wherein the polypeptide is able to stimulate tyrosine
phosphorylation of the p185.sup.HER2 receptor in MDA-MB-453
cells.
31. An isolated polynucleotide encoding a polypeptide comprising
the amino acid sequence of heregulin 2-alpha of SEQ ID NO:11 or a
naturally occurring variant thereof, wherein the polynucleotide
encoding said naturally occurring variant specifically hybridizes
under stringent conditions with the complement of a polynucleotide
encoding the growth factor domain (GFD) of heregulin 2-alpha within
SEQ ID NO:11.
32. The polynucleotide of claim 31 which encodes said naturally
occurring variant.
33. A recombinant expression vector comprising the polynucleotide
of claim 31.
34. A host cell comprising the polynucleotide of claim 31.
35. A method for making a polypeptide comprising:
(a) culturing a host cell comprising the polynucleotide of claim 31
such that the polynucleotide is expressed; and
(b) recovering the polypeptide from the cell culture.
36. An isolated polynucleotide encoding a polypeptide comprising
the amino acid sequence of mature heregulin 2-alpha within SEQ ID
NO:11 or an alternative splice variant thereof, wherein the
polynucleotide encoding said alternative splice variant
specifically hybridizes under stringent conditions with the
complement of a polynucleotide encoding the growth factor domain
(GFD) of heregulin 2-alpha within SEQ ID NO:11.
37. The isolated polynucleotide of claim 36 which encodes said
alternative splice variant.
38. A recombinant expression vector comprising the polynucleotide
of claim 36.
39. A host cell comprising the polynucleotide of claim 36.
40. A method for making a polypeptide comprising:
(a) culturing a host cell comprising the polynucleotide of claim 36
such that the polynucleotide is expressed; and
(b) recovering the polypeptide from the cell culture.
Description
DESCRIPTION OF BACKGROUND AND RELATED ART
Cellular protooncogenes encode proteins that are thought to
regulate normal cellular proliferation and differentiation.
Alterations in their structure or amplification of their expression
lead to abnormal cellular growth and have been associated with
carcinogenesis (Bishop J. M., Science 235:305-311, 1987); (Rhims J.
S., Cancer Detection and Prevention 11:139-149, 1988); (Nowell P.
C., Cancer Res 46:2203-2207, 1986); (Nicolson G. L., Cancer
Res47:1473-1487, 1987). Protooncogenes were first identified by
either of two approaches. First, molecular characterization of the
genomes of transforming retroviruses showed that the genes
responsible for the transforming ability of the virus in many cases
were altered versions of genes found in the genomes of normal
cells. The normal version is the protooncogene, which is altered by
mutation to give rise to the oncogene. An example of such a gene
pair is represented by the EGF receptor and the v-erB gene product.
The virally encoded v-erB gene product has suffered truncation and
other alterations that render it constitutively active and endow it
with the ability to induce cellular transformation (Yarden Y.,
Ullrich A. L., Ann Rev Biochem 57:443-478, 1988).
The second method for detecting cellular transforming genes that
behave in a dominant fashion involves transfection of cellular DNA
from tumor cells of various species into nontransformed target
cells of a heterologous species. Most often this was done by
transfection of human, avian, or rat DNAs into the murine NIH 3T3
cell line (Bishop J. M., Science 235:305-311, 1987); (Rhims J. S.,
Cancer Detection and Prevention 11:139-149, 1988); (Nowell P. C.,
Cancer Res 46:2203-2207, 1986); (Nicolson G. L., Cancer Res
47:1473-1487, 1987); (Yarden Y., Ullrich A. L., Ann Rev Biochem
57:443-478, 1988). Following several cycles of genomic DNA
isolation and retransfection, the human or other species DNA was
molecularly cloned from the murine background and subsequently
characterized. In some cases, the same genes were isolated
following transfection and cloning as those identified by the
direct characterization of transforming viruses. In other cases,
novel oncogenes were identified. An example of a novel oncogene
identified by this transfection assay is the neu oncogene. It was
discovered by Weinberg and colleagues in a transfection experiment
in which the initial DNA was derived from a carcinogen-induced rat
neuroblastoma (Padhy L. et al., Cell 28:865-871, 1982.); (Schechter
A. L. et al., Nature 312:513-516, 1984.) Characterization of the
rat neu oncogene revealed that it had the structure of a growth
factor receptor tyrosine kinase, had homology to the EGF receptor,
and differed from its normal counterpart, the neu protooncogene, by
an activating mutation in its transmembrane domain (Bargmann C. I.,
Hung M. C., Weinberg R. A., Cell45:649-657, 1986). The human
counterpart to neu is the HER2 protooncogene, also designated
c-erbB2 (Coussens et al., Science, 230:1137-1139, 1985; U.S. Ser.
No. 07/143,912 now abandoned).
The association of the HER2 protooncogene with cancer was
established by yet a third approach, that is, its association with
human breast cancer. The HER2 protooncogene was first discovered in
cDNA libraries by virtue of its homology with the EGF receptor,
with which it shares structural similarities throughout (Yarden Y.,
Ullrich A. L. , Ann Rev Biochem 57:443-478, 1988). When radioactive
probes derived from the cDNA sequence encoding p185 HER2 were used
to screen DNA samples from breast cancer patients, amplification of
the HER2 protooncogene was observed in about 30% of the patient
samples (Slamon D. J., Clark G. M., Wong S. G., Levin W. J.,
Ullrich A., McGuire W. L., Science 235:177-182, 1987). Further
studies have confirmed this original observation and extended it to
suggest an important correlation between HER2 protooncogene
amplification and/or overexpression and worsened prognosis in
ovarian cancer and non-small cell lung cancer (Slamon D. J., et
al., Science 244:707-712, 1989); (Wright C., et al., Cancer Res
49:2087-2090, 1989); (Paik S., et al., J Clin Oncology 8:103-112,
1990); (Berchuck A., et al., Cancer Res. 50:4087-4091, 1990); (Kern
J. A., et al., Cancer Res. 50:5184-5191, 1990).
The association of HER2 amplification/overexpression with
aggressive malignancy, as described above, implies that it may have
an important role in progression of human cancer; however, many
tumor-related cell surface antigens have been described in the
past, few of which appear to have a direct role in the genesis or
progression of disease (Schlom J., et al. Cancer Res 50:820-827,
1990); (Szala S., et al., Proc. Natl. Acad Sci. 98:3542-3546).
Among the protooncogenes are those that encode cellular growth
factors which act through endoplasmic kinase phosphorylation of
cytoplasmic protein. The HER1 gene (or ERB-B1) encodes the
epidermal growth factor (EGF) receptor. The .beta.-chain of
platelet-derived growth factor is encoded by the c-sis gene. The
granulocyte-macrophage colony stimulating factor is encoded by the
c-fms gene. The neu proto-oncogene has been identified in
ethylnitrosourea-induced rat neuroblastomas.
The known receptor tyrosine kinases all have the same general
structural motif: an extracellular domain that binds ligand, and an
intracellular tyrosine kinase domain that is necessary for signal
transduction and transformation. These two domains are connected by
a single stretch of approximately 20 mostly hydrophobic amino
acids, called the transmembrane spanning sequence. This
transmembrane spanning sequence is thought to play a role in
transferring the signal generated by ligand binding from the
outside of the cell to the inside. Consistent with this general
structure, the human p185.sup.HER2 glycoprotein, which is located
on the cell surface, may be divided into three principal portions:
an extracellular domain, or ECD (also known as XCD); a
transmembrane spanning sequence; and a cytoplasmic, intracellular
tyrosine kinase domain. While it is presumed that the extracellular
domain is a ligand receptor, the p185.sup.HER2 ligand has not yet
been positively identified. The HER2 gene encodes the 1,255 amino
acid tyrosine kinase receptor-like glycoprotein p185.sup.HER2 that
has homology to the human epidermal growth factor receptor. No
specific ligand binding to p185.sup.HER2 has been identified,
although Lupu et al. (Science 249:1552-1555, 1989) describe an
inhibitory 30 kDa glycoprotein secreted from human breast cancer
cells which is alleged to be a putative ligand for p185.sup.HER2
Lupu et al. (Proceedings of the American Assoc for Cancer Research,
Vol 32, Abs 297, March 1991) reported the purification of a 30 kDa
factor from MDA-MB-231 cells and a 75 kDa factor from SK-BR-3 cells
that stimulates p185.sup.HER2. The 75 kDa factor reportedly induced
phosphorylation of p186.sup.HER2 and modulated cell proliferation
and colony formation of SK-BR-3 cells overexpressing the
p186.sup.HER2 receptor. In the rat neu system, Yarden et al.
(Biochemistry, 30:3543-3550, 1991) describes a 35 kDa glycoprotein
candidate ligand for the neu encoded receptor secreted by ras
transformed fibroblasts.
Methods for the in vivo assay of tumors using HER2 specific
monoclonal antibodies and methods of treating tumor cells using
HER2 specific monoclonal antibodies are described in U.S. Ser. No.
07/143,912 now abandoned. There is a current and continuing need in
the art to identify the actual ligand or ligands that activate
p185.sup.HER2, and to identify their biological role(s), including
their roles in cell-growth and differentiation, cell-transformation
and the creation of malignant neoplasms. While the role of the
p185.sup.HER2 and its ligands is unknown in normal cell growth and
differentiation, it is an object of the present invention to
develop therapeutic uses for the p185.sup.HER2 ligands of the
present invention in promoting normal growth and development.
Accordingly, it is an object of this invention to identify one or
more novel p185.sup.HER2 ligand polypeptide(s) that bind and
stimulate p185.sup.HER2.
It is another object to provide nucleic acid encoding a novel
p185.sup.HER2 binding ligand polypeptides and to use this nucleic
acid to produce a p185.sup.HER2 binding ligand polypeptide in
recombinant cell culture for therapeutic or diagnostic use, and for
the production of therapeutic antagonists for use in certain
metabolic disorders including, but not necessarily restricted to
the killing, inhibition and/or diagnostic imaging of tumors and
tumorigenic cells.
It is a further object to provide derivatives and modified forms of
novel glycoprotein ligands, including amino acid sequence variants,
fusion polypeptides combining a p185.sup.HER2 binding ligand and a
heterologous protein and covalent derivatives of a p185.sup.HER2
binding ligand.
It is an additional object to prepare immunogens for raising
antibodies against a novel p185.sup.HER2 binding ligand, as well as
to obtain antibodies capable of binding to such ligands, and
antibodies which bind a p185.sup.HER2 binding ligand and prevent
the ligand from activating p185.sup.HER2. It is a further object to
prepare immunogens comprising a novel p185.sup.HER2 binding ligand
which is associated with an immunogenic heterologous
polypeptide.
These and other objects of the invention will be apparent to the
ordinary artisan upon consideration of the specification as a
whole.
SUMMARY OF THE INVENTION
In accordance with the objects of this invention, we have
identified and isolated novel ligand families which bind to
p185.sup.HER2. These ligands are denominated the heregulin 2 (HRG2)
polypeptides, and include HRG2-.alpha. and HRG2-.beta.. This
p185.sup.HER2 receptor binding ligand family is hereafter termed
HRG2, or HRG2 variant, and includes N-terminal and C-terminal
fragments thereof. A preferred HRG2 is the 45 kDa ligand disclosed
in FIG. 4 and further designated HRG2-.alpha.. Another preferred
HRG2 is the 14 kDa ligand disclosed in FIG. 8 and designated
HRG2-.beta..
In another aspect, the invention provides a composition comprising
the HRG2 that is free of contaminating human polypeptides. HRG2 or
HRG2 fragments (which also may be synthesized by in vitro methods)
are fused (by recombinant expression or an in vitro peptidyl bond)
to an immunogenic polypeptide and this fusion polypeptide, in turn,
is used to raise antibodies against an HRG2 epitope. Anti-HRG2
antibodies are recovered from the serum of immunized animals.
Alternatively, monoclonal antibodies are prepared from in vitro
cells or in vivo immunized animal in conventional fashion.
Preferred antibodies identified by routine screening will bind to
HRG2, but will not substantially cross-react with any other known
ligands, and will prevent HRG2 from activating p185.sup.HER2.
Immobilized anti-HRG2 antibodies are useful in the diagnosis (in
vitro or in vivo) or purification of the HRG2. In one preferred
embodiment, a mixture of HRG2 and other peptides is passed over a
column to which the anti-HRG2 antibodies are bound.
Substitutional, deletional, or insertional variants of the HRG2 are
prepared by in vitro or recombinant methods and screened for
immuno-crossreactivity with the native forms of HRG2 and for HRG2
antagonist or agonist activity.
In another preferred embodiment, the HRG2 is used as an agonist for
stimulating the activity of p185.sup.HER2. In another preferred
embodiment, a variant of the HRG2 is used as an antagonist to
inhibit stimulation of the p185.sup.HER2.
HRG2 also is derivatized in vitro to prepare immobilized HRG2 and
labeled HRG2, particularly for purposes of diagnosis of HRG2 or its
antibodies, or for affinity purification of HRG2 antibodies.
HRG2, its derivatives, or its antibodies are formulated into
physiologically acceptable vehicles, especially for therapeutic
use. Such vehicles include sustained-release formulations of the
HRG2 or HRG2 variants. A composition is also provided comprising
HRG2 and a pharmaceutically acceptable carrier, and an isolated
polypeptide comprising HRG2 fused to a heterologous
polypeptide.
In still other aspects, the invention provides an isolated nucleic
acid molecule encoding an HRG2, which nucleic acid may be labeled
or unlabeled with a detectable moiety, and a nucleic acid sequence
that is complementary, or hybridizes under stringent conditions to,
a nucleic acid sequence encoding an HRG2.
The nucleic acid sequence is also useful in hybridization assays
for HRG2 nucleic acid and in a method of determining the presence
of an HRG2, comprising hybridizing the DNA (or RNA) encoding (or
complementary to) an HRG2 to a test sample nucleic acid and
determining the presence of an HRG2. The invention also provides a
method of amplifying a nucleic acid test sample comprising priming
a nucleic acid polymerase (chain) reaction with nucleic acid (DNA
or RNA) encoding (or complementary to) a HRG2.
In still further aspects, the nucleic acid molecule is DNA and
further comprises a promoter operably linked to the nucleic acid
sequence.
In addition, the invention provides a replicable vector comprising
the nucleic acid molecule encoding an HRG2 operably linked to
control sequences recognized by a host transformed by the vector;
host cells transformed with the vector; and a method of using a
nucleic acid molecule encoding an HRG2 to effect the production of
HRG2, comprising expressing the nucleic acid molecule in a culture
of the transformed host cells and recovering an HRG2 from the host
cell culture.
In further embodiments, the invention provides a method for
producing HRG2 comprising inserting into the DNA of a cell
containing the nucleic acid encoding an HRG2 a transcription
modulatory element in sufficient proximity and orientation to an
HRG2 nucleic acid to influence transcription thereof, with an
optional further step comprising culturing the cell containing the
transcription modulatory element and an HRG2 nucleic acid.
In still further embodiments, the invention provides a cell
comprising the nucleic acid encoding an HRG2 and an exogenous
transcription modulatory element in sufficient proximity and
orientation to an HRG2 nucleic acid to influence transcription
thereof; and a host cell containing the nucleic acid encoding an
HRG2 operably linked to exogenous control sequences recognized by
the host cell.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Purification of Heregulin-2 on PolyAspartic Acid column
PolyAspartic acid column chromography of heregulin 2-.alpha. was
conducted and the elution profile of proteins measured at
A.sub.214. The 0.6M NaCl pool from the Heparin Sepharose
purification step was diluted to 0.2M NaCl with water and loaded
onto the polyaspartic acid column equilibrated in 17 mM Na
phosphate, pH 6.8 with 30% ethanol. A linear NaCl gradient from 0.3
to 0.6M was initiated at 0 time and was complete at 30 minutes.
Fractions were tested in the HRG2 tyrosine autophosphorylation
assay. The fractions corresponding to peak C were pooled for
further purification on C4 reversed phase HPLC.
FIG. 2. C4 Reversed Phase Purification of Heregulin-2
FIG. 2A: Pool C from the polyaspartic acid column was applied to a
C4 HPLC column (SynChropak RP-4) equilibrated in 0.1% TFA and the
proteins eluted with a linear acetonitrile gradient at
0.25%/minute. The absorbance trace for the run numbered C4-17 is
shown. One milliliter fractions were collected for assay.
FIG. 2B: Ten microliter aliquots of the fractions were tested in
the HRG2 tyrosine autophosphorylation assay. Levels of
phosphotyrosine in the p185.sup.HER2 protein were quantitated by a
specific antiphosphotyrosine antibody and displayed in arbitrary
units on the absissa.
FIG. 2C: Ten microliter fractions were taken and subjected to SDS
gel electrophoresis on 4-20% acrylamide gradient gels according to
the procedure of Laemmli (Laemmli, U.K., Nature, 227:680-685,
1970). The molecular weights of the standard proteins are indicated
to the left of the lane containing the standards. The major peak of
tyrosine phosphorylation activity found in fraction 17 was
associated with a prominent 45,000 Da band (HRG2-.alpha.). Another
peak of activity (fraction 40) was associated with a protein of
apparent molecular weight of 14,000 Da (HRG2-.beta.).
FIG. 3. SDS Polyacrylamide Gel Showing Purification of
Heregulin-2-.alpha.
Molecular weight markers are shown in Lane 1. Aliquots from the
MDA-MB-231 conditioned media (Lane 2), the 0.6M NaCl pool from the
Heparin Sepharose column (Lane 3), Pool C from the polyaspartic
acid column (Lane 4) and Fraction 17 from the HPLC column (C4-17)
(Lane 5) were electrophoresed on a 4-20% gradient gel and silver
stained. Lanes 6 and 7 contained buffer only and shows the presence
of gel artifacts in the 50-65 KDa molecular weight region.
FIGS. 4A to 4D depict the entire coding DNA nucleotide sequence of
the known heregulin 2-.alpha. and the deduced amino acid sequence
of the cDNA contained in .lambda.gt.sub.10 her16 (Seq. ID Nos 10
and 11). The nucleotides are numbered at the top left of each line
and the amino acids written in single letter code are numbered at
the bottom left of each line. The nucleotide sequence corresponding
to the probe is nucleotides 681-720. The probable transmembrane
amino acid domain is amino acids 287-309. The six cysteines of the
EGF motif are 226, 234, 240, 254, and 256. The four potential
three-amino acid N-linked glycosylation sites are 164-166, 170-172,
208-210 and 437-439. The serine-threonine potential O-glycosylation
sites are 209-221. Serine-glycine dipeptide potential
glycosaminoglycan addition sites are amino acids 42-43, 64-65 and
151-152.
FIG. 5. Northern blot analysis of MDA-MB-231 and SKBR3 RNAs
Labeled from left to right are the following: 1) MDA-MB-231 polyA
minus-RNA, (RNA remaining after polyA-containing RNA is removed);
2) MDA-MB-231 polyA plus-mRNA (RNA which contains polyA); 3) SKBR3
polyA minus-RNA; and, 4) SKBR3 polyA plus-mRNA. The probe used for
this analysis was a radioactively (.sup.32 P) labelled internal
xho1 DNA restriction endonuclease fragment from the cDNA portion of
.lambda.gt10her16.
FIG. 6. Sequence Comparisons in the EGF Family of Proteins
Sequences of several EGF-like proteins around the cysteine domain
are aligned with the sequence of HRG2-.alpha.. The location of the
cysteines and the invarient glycine and arginine residues at
positions 238 and 264 clearly show that HRG2-.alpha. is a member of
the EGF family. The region of highest amino acid identity of the
family members relative to HRG2-.alpha. (30-40%) is found between
Cys 236 and Cys 264. The strongest identity (40%) is with the
heparin-binding EGF (HB-EGF) species. HRG2-.alpha. has a unique 3
amino acid insert between Cys 240 and Cys 254. Potential
transmembrane domains are boxed (287-309). Bars indicate the
carboxy-terminal sites for EGF and TGF-alpha where proteolytic
cleavage detaches the mature growth factors from their
transmembrane associated proforms. HB-EGF is heparin
binding-epidermal growth factor; EGF is epidermal growth factor;
TGF-alpha is transforming growth factor alpha; and schwannoma is
the schwannoma-derived growth factor.
FIG. 7. Stimulation of Cell Growth by HRG2-.alpha.
Three different cell lines were tested for growth responses to 1 nM
HRG2-.alpha.. Cell protein was quantitated by crystal violet
staining and the responses normalized to control, untreated
cells.
FIG. 8. Sequence of 14 KDa Protein (HRG2-.beta.)
The N-terminal amino acid sequence (Seq. ID #7) of the protein in
fraction 40 which displays marked p185.sup.HER2 autophosphorylation
activity (see FIG. 2) was determined by conventional Edman
degradation techniques. Residues which could not be determined from
the sequencer data are represented with an X, while tentative
residues are in brackets.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Described is the amino acid sequence and the DNA sequence encoding
heregulin 2 receptor (HRG2) binding ligands. These ligands have
affinity for and stimulate p185.sup.HER2 in autophosphorylation.
Included within the definition of HRG2, in addition to HRG2-.alpha.
and HRG2-.beta., are other polypeptides binding to the HER2 encoded
receptor, which bear substantial amino acid sequence homology to
HRG2-.alpha. or HRG2-.beta., except in the case of HRG2-.alpha.,
that known members of the EGF family as set forth in FIG. 6 are
excluded. Such additional polypeptides fall within the definition
of HRG2 as a family of polypeptide ligands that bind to the HER2
encoded receptor p185.sup.HER2.
I. Definitions
In general, the following words or phrases have the indicated
definition when used in the description, examples, and claims.
Heregulin 2-.alpha.
Heregulin 2-.alpha. (HRG2-.alpha.) is defined herein to be any
isolated polypeptide sequence which possesses a biological property
of a naturally occurring polypeptide comprising the polypeptide
sequence of FIG. 4.
"Biological property" for the purposes herein means an in vivo
effector or antigenic function or activity that is directly or
indirectly performed by the FIG. 4 sequence (whether in its native
or denatured conformation), or by any subsequence thereof. Effector
functions include receptor binding, any enzyme activity or enzyme
modulatory activity, any carrier binding activity, any hormonal
activity, any activity in promoting or inhibiting adhesion of cells
to extracellular matrix or cell surface molecules, or any
structural role. However, effector functions do not include
possession of an epitope or antigenic site that is capable of
cross-reacting with antibodies raised against a polypeptide
sequence of FIG. 4. An antigenic function means possession of an
epitope or antigenic site that is capable of cross-reacting with
antibodies raised against a polypeptide sequence of FIG. 4.
Biologically active HRG2-.alpha. is defined herein as a polypeptide
sharing an effector function of FIG. 4 HRG2-.alpha. and which may
(but need not) in addition possess an antigenic function. A
principal known effector function of HRG2-.alpha. is as a ligand
polypeptide having a qualitative biological activity of binding to
p185.sup.HER2 resulting in the activation of the receptor tyrosine
kinase. Included within the scope of the HRG2-.alpha. as that term
is used herein are HRG2-.alpha. having translated mature amino acid
sequence of the human HRG2-.alpha. as set forth in FIG. 4,
deglycosylated or unglycosylated derivatives of the HRG2-.alpha.,
homologous amino acid sequence variants of the sequence of FIG. 4,
and homologous in vitro-generated variants and derivatives of the
HRG2-.alpha., which are capable of exhibiting a biological activity
in common with the HRG2-.alpha. of FIG. 4. While native
HRG2-.alpha. is a membrane-bound polypeptide, soluble forms, such
as those forms lacking a functional transmembrane domain, are also
included within this definition. In particular, included are
polypeptide fragments of the FIG. 4 prosequence which have an
N-terminal at any residue from about S216 to A227, and its
C-terminus at any residue about from K272 to R286, hereinafter the
growth factor domain (GFD). For purposes of brevity, reference
hereinafter to FIG. 4 and HRG2-.alpha. shall be read as reference
to the GFD fragment.
Antigenically active HRG2-.alpha. is defined as a polypeptide that
possesses an antigenic function of FIG. 4 HRG2-.alpha. and which
may (but need not) in addition possess an effector function.
In preferred embodiments, antigenically active HRG2-.alpha. is a
polypeptide that binds with an affinity of at least about 10.sup.-9
l/mole to an antibody raised against the sequence of FIG. 4.
Ordinarily the polypeptide binds with an affinity of at least about
10.sup.-8 l/mole. Most preferably, the antigenically active
HRG2-.alpha. is a polypeptide that binds to an antibody raised
against the FIG. 4 HRG2-.alpha. in its native conformation. FIG. 4
HRG2-.alpha. in its native conformation is HRG2-.alpha. as found in
nature which has not been denatured by chaotropic agents, heat or
other treatment that substantially modifies the three dimensional
structure of HRG2-.alpha. as determined, for example, by migration
on nonreducing, nondenaturing sizing gels. Antibody used in this
determination is rabbit polyclonal antibody raised by formulating
native HRG2-.alpha. from a non-rabbit species in Freund's complete
adjuvant, subcutaneously injecting the formulation, and boosting
the immune response by intraperitoneal injection of the formulation
until the titer of anti-HRG2-.alpha. antibody plateaus.
Ordinarily, biologically or antigenically active HRG2-.alpha. will
have an amino acid sequence having at least 75% amino acid sequence
identity with the translated HRG2-.alpha. sequence shown in FIG. 4,
more preferably at least 80%, even more preferably at least 90%,
and most preferably at least 95%. Identity or homology with respect
to the FIG. 4 sequence is defined herein as the percentage of amino
acid residues in the candidate sequence that are identical with the
residues in FIG. 4, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent homology, and
not considering any conservative substitutions as part of the
sequence identity. None of N-terminal, C-terminal or internal
extensions, deletions, or insertions into the FIG. 4 sequence shall
be construed as affecting homology.
Thus, the biologically active and antigenically active HRG2-.alpha.
polypeptides that are the subject of this invention include the
sequence of the entire translated nucleotide sequence of FIG. 4;
the mature HRG2-.alpha. of FIG. 4; fragments thereof having a
consecutive sequence of at least 5, 10, 15, 20, 25, 30 or 40 amino
acid residues from the FIG. 4 sequence; amino acid sequence
variants of the FIG. 4 sequence wherein an amino acid residue has
been inserted N- or C-terminal to, or within, the FIG. 4 sequence
or its fragment as defined above; amino acid sequence variants of
the FIG. 4 sequence or its fragment as defined above wherein an
amino acid residue of the FIG. 4 sequence or its fragment as
defined above wherein an amino acid residue of the FIG. 4 sequence
or fragment thereof has been substituted by another residue,
including predetermined mutations by, e.g., site-directed or PCR
mutagenesis, and other animal species of HRG2-.alpha. polypeptides
such as rabbit, rat, porcine, non-human primate, equine, murine,
and ovine HRG2-.alpha. and alleles or other naturally occurring
variants of the foregoing and human sequences; derivatives of
HRG2-.alpha. or its fragments as defined above wherein HRG2-.alpha.
or its fragments have been covalent modified by substitution,
chemical, enzymatic, or other appropriate means, with a moiety
other than a naturally occurring amino acid; glycosylation variants
of HRG2-.alpha. (insertion of a glycosylation site or deletion of
any glycosylation site by deletion, insertion or substitution of
suitable residues); and soluble forms of the HRG2-.alpha., such as
those that lack a functional transmembrane domain. Such fragments
and variants exclude any polypeptide heretofore identified,
including any known protein or polypeptide of any animal species
fragment, which is otherwise anticipatory under 35 U.S.C. 102 as
well as polypeptides obvious over such known protein or
polypeptides under 35 U.S.C. 103.
"Isolated" HRG2-.alpha. means HRG2-.alpha. which has been
identified and separated and/or recovered from a component of its
natural environment. Contaminant components of its natural
environment are materials which would interfere with diagnostic or
therapeutic uses for HRG2-.alpha., and may include proteins,
hormones, and other substances. In preferred embodiments,
HRG2-.alpha. will be purified (1) to greater than 95% by weight of
protein as determined by the Lowry method or other validated
protein determination method, and most preferably more than 99% by
weight, (2) to a degree sufficient to obtain at least 15 residues
of N-terminal or internal amino acid sequence by use of an amino
acid sequenator, or (3) to homogeneity by SDS-PAGE using Coomassie
blue or, preferably, silver stain. Isolated HRG2-.alpha. includes
HRG2-.alpha. in situ within recombinant cells since at least one
component of the HRG2-.alpha. natural environment will not be
present. Ordinarily, however, isolated HRG2-.alpha. will be
prepared by at least one purification step.
Identity or homology with respect to a HRG2-.alpha. is defined
herein as the percentage of amino acid residues in the candidate
sequence that are identical with the residues in FIG. 4, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent homology, and not considering any
conservative substitutions as part of the sequence identity. No N-
nor C-terminal extensions, deletions nor insertions shall be
construed as reducing identity or homology.
In accordance with this invention, HRG2-.alpha. nucleic acid is RNA
or DNA containing greater than ten bases that encodes a
biologically or antigenically active HRG2-.alpha., is complementary
to nucleic acid sequence encoding such HRG2-.alpha., or hybridizes
to nucleic acid sequence encoding such HRG2-.alpha. and remains
stably bound to it under stringent conditions.
Preferably, the HRG2-.alpha. nucleic acid encodes a polypeptide
sharing at least 75% sequence identity, more preferably at least
80%, still more preferably at least 85%, even more preferably at
90%, and most preferably 95%, with the translated amino acid
sequence shown in FIG. 4. Preferably, the HRG2-.alpha. nucleic acid
molecule that hybridizes to the nucleic acid sequence of FIG. 4
contains at least 20, more preferably 40, and most preferably 90
bases. Such hybridizing or complementary nucleic acid, however, is
further defined as being novel under 35 U.S.C. 102 and unobvious
under 35 U.S.C. 103 over any prior art nucleic acid.
Isolated HRG2-.alpha. nucleic acid is a nucleic acid that is
identified and separated from at least one contaminant nucleic acid
with which it is ordinarily associated in the natural source of the
HRG2-.alpha. nucleic acid. Isolated HRG2-.alpha. nucleic acid is
other than in the form or setting in which it is found in nature.
Isolated HRG2-.alpha. nucleic acid therefore distinguishes
HRG2-.alpha. nucleic acid as it exists in natural cells. However,
isolated HRG2-.alpha. encoding nucleic acid includes HRG2-.alpha.
nucleic acid in ordinarily HRG2-.alpha.-expressing cells where the
nucleic acid is, for example, in a chromosomal location different
from that of natural cells.
Heregulin 2-.beta.
HRG2-.beta. is defined herein to be any polypeptide sequence which
possesses a biological property of a naturally occurring
polypeptide comprising the polypeptide sequence of FIG. 8.
"Biological property" for the purposes herein means an in vivo
effector or antigenic function or activity that is directly or
indirectly performed by the HDG2-.beta. (whether in its native or
denatured conformation), or by any subsequence thereof. Effector
functions include receptor binding, any enzyme activity or enzyme
modulatory activity, any carrier binding activity, any hormonal
activity, any activity in promoting or inhibiting adhesion of cells
to extracellular matrix or cell surface molecules, or any
structural role. However, effector functions do not include
possession of an epitope or antigenic site that is capable of
cross-reacting with antibodies raised against a polypeptide
sequence of HRG2-.beta.. An antigen function means possession of an
epitope or antigenic site that is capable of crossreacting with
antibodies raised against a polypeptide sequence of
HRG2-.beta..
Biologically active HRG2-.beta. is defined herein as a polypeptide
sharing an effector function of HRG2-.beta. and which may (but need
not) in addition possess an antigenic function. A principal known
effect or function of HRG2-.beta. is as a ligand polypeptide
binding to p185.sup.HER2 and which has at least 75% amino acid
sequence identity with HRG2-.beta.. Included within the scope of
the biologically active HRG2-.beta. as that term is used herein are
HRG2-.beta. having translated mature amino acid sequences of the
human HRG2-.beta., deglycosylated or unglycosylated derivatives of
the HRG2-.beta., homologous amino acid sequence variants of the
sequence of HRG2-.beta., and homologous in vitro-generated variants
and derivatives of the HRG2-.beta., which are capable of exhibiting
a biological activity in common with the HRG2-.beta.. Also included
within the term HRG2-.beta. are fragments thereof having at least
15 and preferably at least 25 amino acid residues; fragments
thereof having greater than about 5 residues comprising an immune
epitope or other biologically active site of the HRG2-.beta.; amino
acid sequence variants of HRG2-.beta. sequence wherein an amino
acid residue has been inserted N- or C-terminal to, or within, the
HRG2-.beta. sequence or its fragments as defined above; and/or
amino acid sequence variants of said sequence or its fragment as
defined above wherein an amino acid residue of said HRG2-.beta.
sequence or fragment thereof has been substituted by another
residue. This includes predetermined mutations by, e.g.,
site-directed or PCR mutagenesis of an HRG2-.beta. protein from
other animal species such as rabbit, rat, porcine, non-human
primate, equine, murine, and ovine; HRG2-.beta. variants and
alleles and other naturally occurring variants of the foregoing and
human sequences; and derivatives of the HRG2-.beta. or its
fragments as defined above wherein the HRG2-.beta. or its fragments
have been covalently modified by substitution, chemical, enzymatic,
or other appropriate means, with a moiety other than a naturally
occurring amino acid. Such HRG2-.beta. and variants exclude any
polypeptide heretofore identified, which would anticipate the
HRG2-.beta. under 25 USC 102 or make the same obvious under 35 USC
103. HRG2-.beta. amino acid sequence variants generally will share
at least about 80%, more preferably >85% sequence identity with
the HRG2-.beta.
Antigenically active HRG2-.beta. is defined as a polypeptide that
possesses an antigenic function of HRG2-.beta. and which may (but
need not) in addition possess an effector function.
In preferred embodiments, antigenically active HRG2-.beta. is a
polypeptide that binds with an affinity of at least about 10.sup.-9
l/mole to an antibody raised against a HRG2-.beta. sequence.
Ordinarily the polypeptide binds with an affinity of at least about
10.sup.-8 l/mole. Most preferably, the antigenically active
HRG2-.beta. is a polypeptide that binds to an antibody raised
against HRG2-.beta. in its native conformation. HRG2-.beta. in its
native conformation is HRG2-.beta. as found in nature which has not
been denatured by chaotropic agents, heat or other treatment that
substantially modifies the three dimensional structure of
HRG2-.beta. as determined, for example, by migration on
nonreducing, nondenaturing sizing gels. Antibody used in this
determination is rabbit polyclonal antibody raised by formulating
native HRG2-.beta. from a non-rabbit species in Freund's complete
adjuvant, subcutaneously injecting the formulation, and boosting
the immune response by intraperitoneal injection of the formulation
until the titer of anti-HRG2-.beta. antibody plateaus.
Ordinarily, biologically or antigenically active HRG2-.beta. will
have an amino acid sequence having at least 75% amino acid sequence
identity with the translated HRG2-.beta. sequence shown in FIG. 8,
more preferably at least 80%, even more preferably at least 90%,
and most preferably at least 95%. Identity or homology with respect
to the HRG2-.beta. sequence is defined herein as the percentage of
amino acid residues in the candidate sequence that are identical
with the residues in HRG2-.beta.. After aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology, and not considering any conservative substitutions as
part of the sequence identity. None of N-terminal, C-terminal or
internal extensions, deletions, or insertions into the HRG2-.beta.
sequence shall be construed as affecting homology.
Thus, the biologically active and antigenically active HRG2-.beta.
polypeptides that are the subject of this invention include the
sequence of the entire HRG2-.beta.; the HRG2-.beta. fragment of
FIG. 8; fragments of HRG2-.beta. having a consecutive sequence of
at least 5, 10, 15, 20, or 25 amino acid residues from the
HRG2-.beta. sequence amino acids; additional amino acid sequences
found in naturally occurring HRG2-.beta. adjacent to the FIG. 8
amino acids; amino acid sequence variants of the HRG2-.beta.
sequence wherein an amino acid residue has been inserted N- or
C-terminal to, or within, the HRG2-.beta. sequence or its fragment
as defined above; amino acid sequence variants of the HRG2-.beta.
sequence or its fragment as defined above, wherein an amino acid
residue of the HRG2-.beta. sequence or fragment thereof has been
substituted by another residue, including predetermined mutations
by, e.g., site-directed or PCR mutagenesis, and other animal
species of HRG2-.beta.-like ligands such as rabbit, rat, porcine,
non-human primate, equine, murine, and ovine HRG2-.beta. and
alleles or other naturally occurring variants of the foregoing and
human sequences; derivatives of HRG2-.beta. or its fragments as
defined above wherein HRG2-.beta. or its fragments have been
covalent modified by substitution, chemical, enzymatic, or other
appropriate means, with a moiety other than a naturally occurring
amino acid; glycosylation variants of HRG2-.beta. (insertion of a
glycosylation site or deletion of any glycosylation site by
deletion, insertion or substitution of suitable residues); and
soluble forms of the HRG2-.beta., such as those that lack a
functional transmembrane domain. Such fragments and variants
exclude any polypeptide heretofore identified, including any known
HRG2-.beta. of any animal species or any known polypeptide fragment
which are anticipatory order 35 U.S.C. 102, as well as polypeptides
obvious thereover under 35 U.S.C. 103.
"Isolated" HRG2-.beta. means HRG2-.beta. which has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for HRG2-.beta., and may include proteins, hormones, and other
substances. In preferred embodiments, HRG2-.beta., will be purified
(1) to greater than 95% by weight of protein as determined by the
Lowry method or other validated protein determination method, and
most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of an applied biosystems model 470A vapor phase
sequenator, or (3) to homogeneity by SDS-PAGE using Coomassie Blue
or, preferably, silver stain. Ordinarily, isolated HRG2-.beta. will
be prepared by at least one purification step.
In accordance with this invention, HRG2-.beta. nucleic acid is RNA
or DNA containing greater than ten bases that encodes a
biologically or antigenically active HRG2-.beta. , is complementary
to nucleic acid sequence encoding such HRG2-.beta., or hybridizes
to nucleic acid sequence encoding such HRG2-.beta. and remains
stably bound to it under stringent conditions. Included within the
scope of the term HRG2-.beta. is a polypeptide or polypeptide
variant encoded by an HRG2-.beta. encoding nucleotide sequence.
This HRG2-.beta. encoding nucleotide sequence is determined by
using the amino acid sequence of FIG. 8 to synthesize a DNA probe
and selecting by hybridization cDNA from MDA-MB-231, or other
similar HRG2-.beta. containing cells, using the methods described
in the examples for isolating the cDNA encoding the
HRG2-.alpha..
Preferably, the HRG2-.beta. nucleic acid encodes a polypeptide
sharing at least 75% sequence identity, more preferably at least
80%, still more preferably at least 85%, even more preferably at
90%, and most preferably 95%, with the amino acid sequence of
HRG2-.beta.. Preferably, the HRG2-.beta. nucleic acid molecule that
hybridizes to a nucleic acid sequence encoding the amino acid
sequence of HRG2-.beta. contains at least 20, more preferably 40,
and most preferably 90 bases. Such hybridizing or complementary
nucleic acid, however, is further defined as being novel under 35
U.S.C. 102, and unobvious under 35 U.S.C. 103 over any prior art
nucleic acid.
Isolated HRG2-.beta. nucleic acid is a nucleic acid that is
identified and separated or from at least one contaminent nucleic
acid with which it is ordinarily associated in the natural source
of the HRG2-.beta. nucleic acid. Isolated HRG2-.beta. nucleic acid
is other than in the form or setting in which it is found in
nature. Isolated HRG2-.beta. nucleic acid therefore distinguishes
HRG2-.beta. nucleic acid as it exists in natural cells. However,
isolated HRG2-.beta. encoding nucleic acid includes HRG2-.beta. in
ordinarily HRG2-.beta.-expressing cells where the nucleic acid is,
for example, in a chromosomal location different from that of
natural cells.
"Stringent conditions" are those that (1) employ low ionic strength
and high temperature for washing, for example, 0.015M NACl/0.0015M
sodium citrate/0/1% NaDodSO.sub.4 at 50.degree. C.; (2) employ
during hybridization a denaturing agent such as formamide, for
example, 50% (vol/vol) formamide with 0.1% bovine serum albumin,
0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate
buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at
42.degree. C.; or (3) employ 50% formamide, 5.times.SSC (0.75M
NaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%
sodium pyrophosphate, 5.times.Denhardt's solution, sonicated salmon
sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at
42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC and
0.1% SDS.
The term "control sequences" refers to DNA sequences necessary for
the expression of an operably linked coding sequence in a
particular host organism. The control sequences that are suitable
for prokaryotes, for example, include a promoter, optionally an
operator sequence, a ribosome binding site, and possibly, other as
yet poorly understood sequences. Eukaryotic cells are known to
utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. However
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, then synthetic oligonucleotide adaptors or linkers are used
in accord with conventional practice.
An "exogenous" element is defined herein to mean nucleic acid
sequence that is foreign to the cell, or homologous to the cell but
in a position within the host cell nucleic acid in which the
element is ordinarily not found.
As used herein, the expressions "cell", "cell line", and "cell
culture" are used interchangeably, and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. It will be clear
from the context where distinct designations are intended.
"Plasmids" are designated by a lower case "p" preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are commercially available, are publicly available on an
unrestricted basis, or can be constructed from such available
plasmids in accord with published procedures. In addition, other
equivalent plasmids are known in the art and will be apparent to
the ordinary artisan.
"Restriction Enzyme Digestion" of DNA refers to catalytic cleavage
of the DNA with an enzyme that acts only at certain locations in
the DNA. Such enzymes are called restriction endonucleases, and the
sites for which each is specific is called a restriction site. The
various restriction enzymes used herein are commercially available
and their reaction conditions, cofactors, and other requirements as
established by the enzyme suppliers are used. Restriction enzymes
commonly are designated by abbreviations composed of a capital
letter followed by other letters representing the microorganism
from which each restriction enzyme originally was obtained, and
then a number designating the particular enzyme. In general, about
1 .mu.g of plasmid or DNA fragment is used with about 1-2 units of
enzyme in about 20 .mu.l of buffer solution. Appropriate buffers
and substrate amounts for particular restriction enzymes are
specified by the manufacturer. Incubation of about 1 hour at
37.degree. C. is ordinarily used, but may vary in accordance with
the supplier's instructions. After incubation, protein or
polypeptide is removed by extraction with phenol and chloroform,
and the digested nucleic acid is recovered from the aqueous
fraction by precipitation with ethanol. Digestion with a
restriction enzyme may be followed with bacterial alkaline
phosphatase hydrolysis of the terminal 5' phosphates to prevent the
two restriction cleaved ends of a DNA fragment from "circularizing"
or forming a closed loop that would impede insertion of another DNA
fragment at the restriction site. Unless otherwise stated,
digestion of plasmids is not followed by 5' terminal
dephosphorylation. Procedures and reagents for dephosphorylation
are conventional as described in sections 1.56-1.61 of Sambrook et
al. (Molecular Cloning: A Laboratory Manual New York: Cold Spring
Harbor Laboratory Press, 1989).
"Recovery" or "isolation" of a given fragment of DNA from a
restriction digest means separation of the digest on polyacrylamide
or agarose gel by electrophoresis, identification of the fragment
of interest by comparison of its mobility versus that of marker DNA
fragments of known molecular weight, removal of the gel section
containing the desired fragment, and separation of the gel from
DNA. This procedure is known generally. For example, see Lawn et
al., Nucleic Acids Res., 9:6103-6114 (1981), and Goeddel et al.,
Nucleic Acids Res. 8:4057 (1980).
"Northern analysis" is a method used to identify RNA sequences that
hybridize to a known probe such as an oligonuceiotide, DNA
fragment, cDNA or fragment thereof, or RNA fragment. The probe is
labeled with a radioisotope such as .sup.32 P, or by biotinylation,
or with an enzyme. The RNA to be analyzed is usually
electrophoretically separated on an agarose or polyacrylamide gel,
transferred to nitrocellulose, nylon, or other suitable membrane,
and hybridized with the probe, using standard techniques well known
in the art such as those described in stions 7.39-7.52 of Sambrook
et al., supra.
"Ligation" refers to the process of forming phosphodiester bonds
between two nucleic acid fragments. To ligate the DNA fragments
together, the ends of the DNA fragments must be compatible with
each other. In some cases, the ends will be directly compatible
aftet endonuclease digestion. However, it may be necessary to first
convert the staggered ends commonly produced after endonuclease
digestion to blunt ends to make them compatible for ligation. To
blunt the ends, the DNA is treated in a suitable buffer for at
least 15 minutes at 15.degree. C. with about 10 units of the Klenow
fragment of DNA polymerase I or T4 DNA polymerase in the presence
of the four deoxyribonucleotide triphosphates. The DNA is then
purified by phenol-chloroform extraction and ethanol precipitation.
The DNA fragments that are to be ligated together are put in
solution in about equimolar amounts. The solution will also contain
ATP, ligase buffer, and a ligase such as T4 DNA ligase at about 10
units per 0.5 .mu.g of DNA. If the DNA is to be ligated into a
vector, the vector is first linearized by digestion with the
appropriate restriction endonuclease(s). The linearized fragment is
then treated with bacterial alkaline phosphatase, or calf
intestinal phosphatase to prevent self-ligation during the ligation
step.
"Preparation" of DNA from cells means isolating the plasmid DNA
from a culture of the host cells. Commonly used methods for DNA
preparation are the large and small-scale plasmid preparations
described in sections 1.25-1.33 of Sambrook et al., supra. After
preparation of the DNA, it can be purified by methods well known in
the art such as that described in section 1.40 of Sambrook et al.,
supra.
"Oligonucleotides" are short-length, single- or double-stranded
polydeoxynucleotides that are chemically synthesized by known
methods (such as phosphotriester, phosphite, or phosphoramidite
chemistry, using solid phase techniques such as described in EP
266,032, published 4 May 1988, or via deoxynucleoside H-phosphonate
intermediates as described by Froehler et al., Nucl. Acids Res.,
14:5399-5407, 1986. They are then purified on polyacrylamide
gels.
The technique of "polymerase chain reaction," or "PCR," as used
herein generally refers to a procedure wherein minute amounts of a
specific piece of nucleic acid, RNA and/or DNA, are amplified as
described in U.S. Pat. No. 4,683,195, issued 28 Jul. 1987.
Generally, sequence information from the ends of the region of
interest or beyond needs to be available, such that oligonucleotide
primers can be designed; these primers will be identical or similar
in sequence to opposite strands of the template to be amplified.
The 5' terminal nucleotides of the two primers may coincide with
the ends of the amplified material. PCR can be used to amplify
specific RNA sequences, specific DNA sequences from total genomic
DNA, and cDNA transcribed from total cellular RNA, bacteriophage or
plasmid sequences, etc. See generally Mullis et al., Cold Spring
Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR
Technology, (Stockton Press, NY, 1989). As used herein, PCR is
considered to be one, but not the only, example of a nucleic acid
polymerase reaction method for amplifying a nucleic acid test
sample, comprising the use of a known nucleic acid (DNA or RNA) as
a primer, and utilizes a nucleic acid polymerase to amplify or
generate a specific piece of nucleic acid or to amplify or generate
a specific piece of nucleic acid which is complementary to a
particular nucleic acid.
The "HRG2 tyrosine autophosphorylation assay" to detect the
presence of HRG2 ligands was used to monitor the purification of a
ligand for the p185.sup.HER2 receptor. This assay is based on the
assumption that a specific ligand for the p185.sup.HER2 receptor
will stimulate autophosphorylation of the receptor, in analogy with
EGF and its stimulation of EGF receptor autophosphorylation.
MDA-MB-453 cells, which contain high levels of p.sub.185.sup.HER2
receptors but negligible levels of human EGF receptors, were
obtained from the American Type Culture Collection, Rockville, Md.
(ATCC No HTB-131) and maintained in tissue culture with 10% fetal
calf serum in DMEM/Hams F12 (1:1) media. For assay, the cells were
trypsinized and plated at 150,000 cells/well in 24 well dishes
(Costar). After incubation with serum containing media overnight,
the cells were placed in serum free media for 2-18 hours before
assay. Test samples of 100 uL aliquots were added to each well. The
cells were incubated for 5-30 minutes (typically 30 min) at
37.degree. C. and the media removed. The cells in each well were
treated with 100 uL SDS gel denaturing buffer (Seprosol, Enpotech,
Inc.) and the plates heated at 100.degree. C. for 5 minutes to
dissolve the cells and denature the proteins. Aliquots from each
well were electrophoresed on 5-20% gradient SDS gels (Novex,
Encinitas, Calif.) according to the manufacturer's directions.
After the dye front reached the bottom of the gel, the
electrophoresis was terminated and a sheet of PVDF membrane
(ProBlott, ABI) was placed on the gel and the proteins transfered
from the gel to the membrane in a blotting chamber (BioRad) at 200
mAmps for 30-60 min. After blotting, the membranes were incubated
with Tris buffered saline containing 0.1% Tween 20 detergent buffer
with 5% BSA for 2-18 hrs to block nonspecific binding, and then
treated with a mouse anti-phosphotyrosine antibody (Upstate
Biological Inc., N.Y.). Subsequently, the membrane blots were
treated with goat anti-mouse antibody conjugated to alkaline
phosphatase. The gels were developed using the ProtoBlot System
from Promega. After drying the membranes, the density of the bands
corresponding to p185.sup.HER2 in each sample lane was quantitated
with a Hewlett Packard ScanJet Plus Scanner attached to a Macintosh
computer. The number of receptors per cell in the MDA-MB-453 cells
is such that under these experimental conditions the p185.sup.HER2
receptor protein is the major protein which is labeled.
"Protein microsequencing" was accomplished based upon the following
procedures. Proteins from the final HPLC step were either sequenced
directly by automated Edman degradation with a model 470A Applied
Biosystems gas phase sequencer equipped with a 120A PTH amino acid
analyzer or sequenced after digestion with various chemicals or
enzymes. PTH amino acids were integrated using the ChromPerfect
data system (Justice Innovations, Palo Alto, Calif.). Sequence
interpretation was performed on a VAX 11/785 Digital Equipment
Corporation computer as described (Henzel, et al., J.
Chromatography, 404:41-52 (1987)). In some cases, aliquots of the
HPLC fractions were electrophoresed on 5-20% SDS polyacrylamide
gels, electrotransferred to a PVDF membrane (ProBlott, ABI, Foster
City, Calif.) and stained with Coomassie Brilliant Blue
(Matsudaira, P., J. Biol. Chem., 262:10035-10038, 1987). The
specific protein was excised from the blot for N terminal
sequencing. To determine internal protein sequences, HPLC fractions
were dried under vacuum (SpeedVac), resuspended in appropriate
buffers, and digested with cyanogen bromide, the lysine-specific
enzyme Lys-C (Wako Chemicals, Richmond, Va.) or Asp-N (Boehringer
Mannheim, Indianapolis, Ind.). After digestion, the resultant
peptides were sequenced as a mixture or were resolved by HPLC on a
C4 column developed with a propanol gradient in 0.1% TFA before
sequencing as described above.
II. Suitable Methods for Practicing the Invention
1. Preparation of Native Hereeulin 2 and Variants
The following description is for HRG2-.alpha., however, similar
methods may be used for the preparation of HRG2-.beta. or any
HRG2.
Summary of Method Using DNA Sequence of FIG. 4
Mammalian expression of HRG2-.alpha. may be achieved in a number of
ways. Once the entire coding sequence of preproheregulin 2-.alpha.
is attained, the complete sequence containing an initiating
methionine, presequence, and prosequence through the stop codon
will be inserted into a mammalian expression vector, such as pRK5,
where under control of a promoter, such as the CMV promoter, the
transmembrane-bound growth factor will be expressed following
transfection into a suitable host cell such as COS7. Natural
proteolytic processing to the mature soluble form will occur in the
cell or in the cell-conditioned medium from where it may be
purified. Alternatively, proteolytic enzymes may be added to the
cell and/or to the conditioned medium to achieve the desired
processing resulting in proteolytic cleavage of the soluble
HRG2-.alpha. ligand from the transmembrane sequence. In the absence
of a complete nucleotide sequence, expression may be achieved by
inserting, using standard molecular biological techniques, a start
codon and heterologous presequence anywhere upstream of the
beginning of the active portion of the molecule (prior to the amino
terminus), and a stop codon anywhere COOH-terminal to the beginning
of the transmembrane domain. The mature HRG2-.alpha. will be
processed by natural or artificial proteolytic digestion in the
cell conditioned medium or after purification. A secreted proform
of the molecule where the stop codon is inserted before the
transmembrane domain may in fact not even require processing to the
mature form to be biologically active.
A. Isolation of DNA Encoding Heregulin 2
The DNA encoding the HRG2-.alpha. may be obtained from any cDNA
library prepared from tissue believed to possess the HRG2-.alpha.
mRNA and to express it at a detectable level. The HRG2-.alpha. gene
may also be obtained from a genomic library. Similar procedures may
be used for the isolation of the HRG2-.beta. encoding gene.
Libraries are screened with probes designed to identify the gene of
interest or the protein encoded by it. For cDNA expression
libraries, suitable probes include monoclonal or polyclonal
antibodies that recognize and specifically bind to the
HRG2-.alpha.; oligonucleotides of about 20-80 bases in length that
encode known or suspected portions of the HRG2-.alpha. cDNA from
the same or different species; and/or complementary or homologous
cDNAs or fragments thereof that encode the same or a similar gene.
Appropriate probes for screening genomic DNA libraries include, but
are not limited to, oligonucleotides; cDNAs or fragments thereof
that encode the same or a similar gene; and/or homologous genomic
DNAs or fragments thereof. Screening the cDNA or genomic library
with the selected probe may be conducted using standard procedures
as described in chapters 10-12 of Sambrook et al., supra.
An alternative means to isolate the gene encoding HRG2-.alpha. is
to use polymerase chain reaction (PCR) methodology as described in
section 14 of Sambrook et al., supra. This method requires the use
of oligonucleotide probes that will hybridize to the HRG2-.alpha..
Strategies for selection of oligonucleotides are described
below.
Another alternative method for obtaining the gene of interest is to
chemically synthesize it using one of the methods described in
Engels et al. (Agnew. Chem. Int. Ed. Engl., 28: 716-734,1989),
specifically incorporated by reference. These methods include
triester, phosphite, phosphoramidite and H-Phosphonate methods, PCR
and other autoprimer methods, and oligonucleotide syntheses on
solid supports. These methods may be used if the entire nucleic
acid sequence of the gene is known, or the sequence of the nucleic
acid complementary to the coding strand is available, or
alternatively, if the target amino acid sequence is known, one may
infer potential nucleic acid sequences using known and preferred
coding residues for each amino acid residue.
A preferred method of practicing this invention is to use carefully
selected oligonucleotide sequences to screen cDNA libraries from
various tissues, preferably human breast, colon, salivary gland,
placental, fetal, brain, and carcinoma cell lines. Other biological
sources of DNA encoding an heregulin 2-like ligand include other
mammals and birds. Among the preferred mammals are members of the
following orders: bovine, ovine, equine, murine, and rodentia.
The oligonucleotide sequences selected as probes should be of
sufficient length and sufficiently unambiguous that false positives
are minimized. The actual nucleotide sequence(s) is usually based
on conserved or highly homologous nucleotide sequences or regions
of HRG2-.alpha.. The oligonucleotides may be degenerate at one or
more positions. The use of degenerate oligonucleotides may be of
particular importance where a library is screened from a species in
which preferential codon usage in that species is not known. The
oligonucleotide must be labeled such that it can be detected upon
hybridization to DNA in the library being screened. The preferred
method of labeling is to use .sup.32 P-labeled ATP with
polynucleotide kinase, as is well known in the art, to radiolabel
the oligonucleotide. However, other methods may be used to label
the oligonucleotide, including, but not limited to, biotinylation
or enzyme labeling.
Of particular interest is the HRG2-.alpha. nucleic acid that
encodes a full-length polypeptide. In some preferred embodiments,
the nucleic acid sequence includes the native HRG2-.alpha. signal
sequence. Nucleic acid having all the protein coding sequence is
obtained by screening selected cDNA or genomic libraries, and, if
necessary, using conventional primer extension procedures as
described in section 7.79 of Sambrook et al., supra, to detect
precursors and processing intermediates of mRNA that may not have
been reverse-transcribed into cDNA.
The HRG2-.alpha. encoding DNA of FIG. 4 may be used to isolate DNA
encoding the analogous ligand from other animal species via
hybridization employing the methods discussed above. The preferred
animals are mammals, particularly bovine, ovine, equine, feline,
canine and rodentia, and more specifically rats, mice and
rabbits.
B. Amino Acid Sequence Variants of the Heregulin 2-.alpha.
Amino acid sequence variants of the HRG2-.alpha. are prepared by
introducing appropriate nucleotide changes into the HRG2-.alpha.
DNA, or by in vitro synthesis of the desired HRG2-.alpha.
polypeptide. Such variants include, for example, deletions from, or
insertions or substitutions of, residues within the amino acid
sequence shown for the human HRG2-.alpha. in FIG. 4. Any
combination of deletion, insertion, and substitution can be made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics. Excluded from the scope of
this invention are HRG2 variants or polypeptide sequences that are
not novel and unobvious over the prior art. The amino acid changes
also may alter post-translational processes of the HRG2-.alpha.,
such as changing the number or position of glycosylation sites,
altering the membrane anchoring characteristics, and/or altering
the intra-cellular location of the HRG2-.alpha. by inserting,
deleting, or otherwise affecting the leader sequence of the native
HRG2-.alpha..
The amino acid sequence of FIG. 4 may be proteolytically processed
to create a number of HRG2-.alpha. fragments which all contain the
amino acid sequence between cysteine 226 and cysteine 265. The
amino terminus of the HRG2-.alpha. fragment may result from the
cleavage of any peptide bond between alanine 1 and cysteine 226,
preferably adjacent to an arginine, lysine, valine, or methionine,
and most preferably between methionine 45 and serine 46. The
carboxy terminus of the HRG2-.alpha. fragment may result from the
cleavage of any peptide bond between cysteine 265, preferably
adjacent to an arginine, lysine, valine, or methionine, and most
preferably between lysine 272 and valine 273, between lysine 278
and alanine 279, or between lysine 285 and arginine 286. The
resulting HRG2-.alpha. ligands resulting from such proteolytic
processing are the preferred ligands.
In designing amino acid sequence variants of HRG2-.alpha., the
location of the mutation site and the nature of the mutation will
depend on the HRG2-.alpha. characteristic(s) to be modified. The
sites for mutation can be modified individually or in series, e.g.,
by (1) substituting first with conservative amino acid choices and
then with more radical selections depending upon the results
achieved, (2) deleting the target residue, or (3) inserting
residues of other receptor ligands adjacent to the located
site.
A useful method for identification of certain residues or regions
of the HRG2-.alpha. polypeptide that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (Science, 244: 1081-1085, 1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
the surrounding aqueous environment in or outside the cell. Those
domains demonstrating functional sensitivity to the substitutions
then are refined by introducing further or other variants at or for
the sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to optimize
the performance of a mutation at a given site, ala scanning or
random mutagenesis may be conducted at the target codon or region
and the expressed HRG2-.alpha. variants are screened for the
optimal combination of desired activity.
There are two principal variables in the construction of amino acid
sequence variants: the location of the mutation site and the nature
of the mutation. These are variants from the FIG. 4 sequence, and
may represent naturally occurring alleles (which will not require
manipulation of the HRG2-.alpha. DNA) or predetermined mutant forms
made by mutating the DNA, either to arrive at an allele or a
variant not found in nature. In general, the location and nature of
the mutation chosen will depend upon the HRG2-.alpha.
characteristic to be modified. Obviously, such variations that, for
example, convert the HRG2-.alpha. into a known receptor ligand, are
not included within the scope of this invention, nor are any other
HRG2-.alpha. variants or polypeptide sequences that are not novel
and unobvious over the prior art.
Amino acid sequence deletions generally range from about 1 to 30
residues, more preferably about 1 to 10 residues, and typically are
contiguous. Deletions may be introduced into regions of low
homology with other receptor ligands to modify the activity of the
HRG2-.alpha.. Deletions from the HRG2-.alpha. in areas of
substantial homology with any other receptor ligands will be more
likely to modify the biological activity of the HRG2-.alpha. more
significantly. The number of consecutive deletions will be selected
so as to preserve the tertiary structure of HRG2-.alpha. in the
affected domain, e.g., cysteine cross-linking, beta-pleated sheet
or alpha helix.
Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions (i.e., insertions within the HRG2
sequence) may range generally from about 1 to 10 residues, more
preferably 1 to 5, and most preferably 1 to 3. Examples of terminal
insertions include the HRG2-.alpha. with an N-terminal methionyl
residue, an artifact of the direct expression of HRG2-.alpha. in
bacterial recombinant cell culture, and fusion of a heterologous
N-terminal signal sequence to the N-terminus of the HRG2-.alpha.
molecule to facilitate the secretion of the mature HRG2-.alpha.
from recombinant host cells. Such signal sequences generally will
be obtained from, and thus homologous to, the intended host cell
species. Suitable sequences include STII or lpp for E. coli, alpha
factor for yeast, and viral signals such as herpes gD for mammalian
cells.
Other insertional variants of the HRG2-.alpha. include the fusion
to the N- or C-terminus of the HRG2-.alpha. of immunogenic
polypeptides, e.g., bacterial polypeptides such as beta-lactamase
or an enzyme encoded by the E. coli trp locus, or yeast protein,
and C-terminal fusions with proteins having a long half-life such
as immunoglobulin constant regions (or other immunoglobulin
regions), albumin, or ferritin, as described in WO 89/02922,
published 6 Apr. 1989.
Another group of variants are amino acid substitution variants.
These variants have at least one amino acid residue in the
HRG2-.alpha. molecule removed and a different residue inserted in
its place. The sites of greatest interest for substitutional
mutagenesis include sites identified as the active site(s) of the
HRG2-.alpha., and sites where the amino acids found in the
HRG2-.alpha. like ligands from various species are substantially
different in terms of side-chain bulk, charge, and/or
hydrophobicity.
Other sites of interest are those in which particular residues of
the HRG2-like ligands obtained from various species are identical.
These positions may be important for the biological activity of the
HRG2-.alpha.. These sites, especially those falling within a
sequence of at least three other identically conserved sites, are
substituted in a relatively conservative manner. Such conservative
substitutions are shown in Table 1 under the heading of "preferred
substitutions". If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 1, or as further described below
in reference to amino acid classes, are introduced and the products
screened.
TABLE 1 ______________________________________ Original Exemplary
Preferred Residue Substitutions Substitutions
______________________________________ Ala (A) val; leu; ile val
Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D)
glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro
pro His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;
leu norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys
(K) arg; gln; asn arg Met (M) leu;. phe; ile leu Phe (F) leu; val;
ile; ala leu Pro (P) gly gly Ser (S) thr thr Thr (T) ser ser Trp
(W) tyr tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;
phe; leu ala; norleucine ______________________________________
Substantial modifications in function or immunological identity of
the HRG2 are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Naturally occurring residues are divided into
groups based on common side chain properties:
1) hydrophobic: norleucine, met, ala, val, leu, ile;
2) neutral hydrophilic: cys, ser, thr;
3) acidic: asp, glu;
4) basic: asn, gln, his, lys, arg;
5) residues that influence chain orientation: gly, pro; and
6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another. Such substituted residues may be
introduced into regions of the HRG2-.alpha. that are homologous
with other receptor ligands, or, more preferably, into the
non-homologous regions of the molecule.
In one embodiment of the invention, it is desirable to inactivate
one or more protease cleavage sites that are present in the
molecule. These sites are identified by inspection of the encoded
amino acid sequence.
Where protease cleavage sites are identified, they are rendered
inactive to proteolytic cleavage by substituting the targeted
residue with another residue, preferably a basic residue such as
glutamine or a hydrophobic residue such as serine; by deleting the
residue; or by inserting a prolyl residue immediately after the
residue.
In another embodiment, any methionyl residue other than the
starting methionyl residue of the signal sequence, or any residue
located within about three residues N- or C-terminal to each such
methionyl residue, is substituted by another residue (preferably in
accord with Table 1) or deleted. Alternatively, about 1-3 residues
are inserted adjacent to such sites.
Any cysteine residues not involved in maintaining the proper
conformation of HRG2-.alpha. also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross-linking.
Sites particularly suited for substitutions, deletions or
insertions, or use as fragments, include, numbered from the
N-terminus of the HRG2-.alpha. of FIG. 4:
1) potential glycosaminoglycan addition sites at the serine-glycine
dipeptides at 42-43, 64-65, 151-152;
2) potential asparagine-linked glycosylation at positions 164, 170,
208 and 437, sites (NDS) 164-166, (NIT) 170-172 and (NTS)
208-210;
3) potential O-glycosylation in a cluster of serine and threonine
at 209-218;
4) cysteines at 226, 234, 240, 254, 256 and 265;
5) transmembrane domain at 287-309;
6) loop 1 delineated by cysteines 226 and 240;
7) loop 2 delineated by cysteines 234 and 254;
8) loop 3 delineated by cysteines 256 and 265; and
9) potential protease processing sites at 2-3, 8-9, 23-24, 33-34,
36-37, 45-46, 48-49, 62-63, 66-67, 86-87, 110-111, 123-124,
134-135, 142-143, 272-273, 278-279 and 285-286;
DNA encoding amino acid sequence variants of the HRG2-.alpha. is
prepared by a variety of methods known in the art. These methods
include, but are not limited to, isolation from a natural source
(in the case of naturally occurring amino acid sequence variants)
or preparation by oligonucleotide-mediated (or site-directed)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an
earlier prepared variant or a non-variant version of the
HRG2-.alpha.. These techniques may utilize HRG2-.alpha. nucleic
acid (DNA or RNA), or nucleic acid complementary to the
HRG2-.alpha. nucleic acid.
Oligonucleotide-mediated mutagenesis is a preferred method for
preparing substitution, deletion, and insertion variants of
HRG2-.alpha. DNA. This technique is well known in the art as
described by Adelman et al., DNA, 2: 183 (1983). Briefly, the
HRG2-.alpha. DNA is altered by hybridizing an oligonucleotide
encoding the desired mutation to a DNA template, where the template
is the single-stranded form of a plasmid or bacteriophage
containing the unaltered or native DNA sequence of the
HRG2-.alpha.. After hybridization, a DNA polymerase is used to
synthesize an entire second complementary strand of the template
that will thus incorporate the oligonucleotide primer, and will
code for the selected alteration in the HRG2-.alpha. DNA.
Generally, oligonucleotides of at least 25 nucleotides in length
are used. An optimal oligonucleotide will have 12 to 15 nucleotides
that are completely complementary to the template on either side of
the nucleotide(s) coding for the mutation. This ensures that the
oligonucleotide will hybridize properly to the single-stranded DNA
template molecule. The oligonucleotides are readily synthesized
using techniques known in the art such as that described by Crea et
al. (Proc. Natl. Acad. Sci. USA, 75: 5765,1978).
Single-stranded DNA template may also be generated by denaturing
double-stranded plasmid (or other) DNA using standard
techniques.
For alteration of the native DNA sequence (to generate amino acid
sequence variants, for example), the oligonucleotide is hybridized
to the single-stranded template under suitable hybridization
conditions. A DNA polymerizing enzyme, usually the Klenow fragment
of DNA polymerase I, is then added to synthesize the complementary
strand of the template using the oligonucleotide as a primer for
synthesis. A heteroduplex molecule is thus formed such that one
strand of DNA encodes the mutated form of the HRG2-.alpha., and the
other strand (the original template) encodes the native, unaltered
sequence of the HRG2-.alpha.. This heteroduplex molecule is then
transformed into a suitable host cell, usually a prokaryote such as
E. coli JM101. After the cells are grown, they are plated onto
agarose plates and screened using the oligonucleotide primer
radiolabeled with .sup.32 P-phosphate to identify the bacterial
colonies that contain the mutated DNA. The mutated region is then
removed and placed in an appropriate vector for protein production,
generally an expression vector of the type typically employed for
transformation of an appropriate host.
The method described immediately above may be modified such that a
homoduplex molecule is created wherein both strands of the plasmid
contain the mutation(s). The modifications are as follows: the
single-stranded oligonucleotide is annealed to the single-stranded
template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTTP), is combined with a modified
thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham Corporation). This mixture is added to the
template-oligonucleotide complex. Upon addition of DNA polymerase
to this mixture, a strand of DNA identical to the template except
for the mutated bases is generated. In addition, this new strand of
DNA will contain dCTP-(aS) instead of dCTP, which serves to protect
it from restriction endonuclease digestion. After the template
strand of the double-stranded heteroduplex is nicked with an
appropriate restriction enzyme, the template strand can be digested
with ExoIII nuclease or another appropriate nuclease past the
region that contains the site(s) to be mutagenized. The reaction is
then stopped to leave a molecule that is only partially
single-stranded. A complete double-stranded DNA homoduplex is then
formed using DNA polymerase in the presence of all four
deoxyribonucleotide triphosphates, ATP, and DNA ligase. This
homoduplex molecule can then be transformed into a suitable host
cell such as E. coli JM101, as described above.
DNA encoding HRG2-.alpha. mutants with more than one amino acid to
be substituted may be generated in one of several ways. If the
amino acids are located close together in the polypeptide chain,
they may be mutated simultaneously using one oligonucleotide that
codes for all of the desired amino acid substitutions. If, however,
the amino acids are located some distance from each other
(separated by more than about ten amino acids), it is more
difficult to generate a single oligonucleotide that encodes all of
the desired changes. Instead, one of two alternative methods may be
employed.
In the first method, a separate oligonucleotide is generated for
each amino acid to be substituted. The oligonucleotides are then
annealed to the single-stranded template DNA simultaneously, and
the second strand of DNA that is synthesized from the template will
encode all of the desired amino acid substitutions.
The alternative method involves two or more rounds of mutagenesis
to produce the desired mutant. The first round is as described for
the single mutants: wild-type DNA is used for the template, an
oligonucleotide encoding the first desired amino acid
substitution(s) is annealed to this template, and the heteroduplex
DNA molecule is then generated. The second round of mutagenesis
utilizes the mutated DNA produced in the first round of mutagenesis
as the template. Thus, this template already contains one or more
mutations. The oligonucleotide encoding the additional desired
amino acid substitution(s) is then annealed to this template, and
the resulting strand of DNA now encodes mutations from both the
first and second rounds of mutagenesis. This resultant DNA can be
used as a template in a third round of mutagenesis, and so on.
PCR mutagenesis is also suitable for making amino acid variants of
HRG2-.alpha.. While the following discussion refers to DNA, it is
understood that the technique also finds application with RNA. The
PCR technique generally refers to the following procedure (see
Erlich, supra, the chapter by R. Higuchi, p. 61-70). When small
amounts of template DNA are used as starting material in a PCR,
primers that differ slightly in sequence from the corresponding
region in a template DNA can be used to generate relatively large
quantities of a specific DNA fragment that differs from the
template sequence only at the positions where the primers differ
from the template. For introduction of a mutation into a plasmid
DNA, one of the primers is designed to overlap the position of the
mutation and to contain the mutation; the sequence of the other
primer must be identical to a stretch of sequence of the opposite
strand of the plasmid, but this sequence can be located anywhere
along the plasmid the sequence of the s however, that the sequence
of the second primer is located within 200 nucleotides from that of
the first, such that in the end the entire amplified region of DNA
bounded by the primers can be easily sequenced. PCR amplification
using a primer pair like the one just described results in a
population of DNA fragments that differ at the position of the
mutation specified by the primer, and possibly at other positions,
as template copying is somewhat error-prone.
If the ratio of template to product material is extremely low, the
vast majority of product DNA fragments incorporate the desired
mutation(s).
This product material is used to replace the corresponding region
in the plasmid that served as PCR template using standard DNA
technology. Mutations at separate positions can be introduced
simultaneously by either using a mutant second primer, or
performing a second PCR with different mutant primers and ligating
the two resulting PCR fragments simultaneously to the vector
fragment in a three (or more)-part ligation.
In a specific example of PCR mutagenesis, template plasmid DNA
(1.mu.g) is linearized by digestion with a restriction endonuclease
that has a unique recognition site in the plasmid DNA outside of
the region to be amplified. Of this material, 100 ng is added to a
PCR mixture containing PCR buffer, which contains the four
deoxynucleotide tri-phosphates and is included in the GeneAmp.RTM.
kits (obtained from Perkin-Elmer Cetus, Norwalk, Conn. and
Emeryville, Calif.), and 25 pmole of each oligonucleotide primer,
to a final volume of 50 .mu.l. The reaction mixture is overlayed
with 35 .mu.l mineral oil. The reaction is denatured for 5 minutes
at 100.degree. C., placed briefly on ice, and then 1 .mu.l Thermus
aquaticus (Taq) DNA polymerase (5 units/.mu.l, purchased from
Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.) is added
below the mineral oil layer. The reaction mixture is then inserted
into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus)
programmed as follows:
2 min. 55.degree. C.,
30 sec. 72.degree. C., then 19 cycles of the following:
30 sec. 94.degree. C.,
30 sec. 55.degree. C., and
30 sec. 72.degree. C.
At the end of the program, the reaction vial is removed from the
thermal cycler and the aqueous phase transferred to a new vial,
extracted with phenol/chloroform (50:50:vol), and ethanol
precipitated, and the DNA is recovered by standard procedures. This
material is subsequently subjected to the appropriate treatments
for insertion into a vector.
Another method for preparing variants, cassette mutagenesis, is
based on the technique described by Wells et al. (Gene, 34:
315,1985). The starting material is the plasmid (or other vector)
comprising the HRG2 DNA to be mutated. The codon(s) in the
HRG2-.alpha. DNA to be mutated are identified. There must be a
unique restriction endonuclease site on each side of the identified
mutation site(s). If no such restriction sites exist, they may be
generated using the above-described oligonucleotide-mediated
mutagenesis method to introduce them at appropriate locations in
the HRG2-.alpha. DNA. After the restriction sites have been
introduced into the plasmid, the plasmid is cut at these sites to
linearize it. A double-stranded oligonucleotide encoding the
sequence of the DNA between the restriction sites but containing
the desired mutation(s) is synthesized using standard procedures.
The two strands are synthesized separately and then hybridized
together using standard techniques. This double-stranded
oligonucleotide is referred to as the cassette. This cassette is
designed to have 3' and 5' ends that are compatible with the ends
of the linearized plasmid, such that it can be directly ligated to
the plasmid. This plasmid now contains the mutated HRG2-.alpha. DNA
sequence.
C. Insertion of DNA into a Cloning Vehicle
The cDNA or genomic DNA encoding native or variant HRG2-.alpha. is
inserted into a replicable vector for further cloning
(amplification of the DNA) or for expression. Many vectors are
available, and selection of the appropriate vector will depend on
1) whether it is to be used for DNA amplification or for DNA
expression, 2) the size of the DNA to be inserted into the vector,
and 3) the host cell to be transformed with the vector. Each vector
contains various components depending on its function
(amplification of DNA or expression of DNA) and the host cell for
which it is compatible. The vector components generally include,
but are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence.
(i) Signal Sequence Component
In general, the signal sequence may be a component of the vector,
or it may be a part of the HRG2-.alpha. DNA that is inserted into
the vector. The native proHRG2-.alpha. DNA encodes a signal
sequence at the amino terminus (5' end of the DNA encoding
HRG2-.alpha.) of the polypeptide that is cleaved during
post-translational processing of the polypeptide to form the mature
HRG2-.alpha. polypeptide. Native HRG2-.alpha. is not, however,
secreted from the cell as it contains a transmembrane domain and a
cytoplasmic region in the carboxyl terminal region of the
polypeptide. Thus, to form a secreted version of HRG2-.alpha. the
carboxyl terminal domain of the molecule, including the
transmembrane domain, is ordinarily deleted. This truncated variant
HRG2-.alpha. polypeptide may be secreted from the cell, provided
that the DNA encoding the truncated variant retains the amino
terminal signal sequence.
The HRG2-.alpha. of this invention may be expressed not only
directly, but also as a fusion with a heterologous polypeptide,
preferably a signal sequence or other polypeptide having a specific
cleavage site at the N- and/or C-terminis of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the HRG2-.alpha. DNA that is
inserted into the vector. Included within the scope of this
invention are HRG2-.alpha. with the native signal sequence deleted
and replaced with a heterologous signal sequence. The heterologous
signal sequence selected should be one that is recognized and
processed, i.e., cleaved by a signal peptidase, by the host cell.
For prokaryotic host cells that do not recognize and process the
native HRG2-.alpha. signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, lpp, or
heat-stable enterotoxin II leaders. For yeast secretion the native
HRG2-.alpha. signal sequence may be substituted by the yeast
invertase, alpha factor, or acid phosphatase leaders. In mammalian
cell expression the native signal sequence is satisfactory,
although other mammalian signal sequences may be suitable.
(ii) Origin of Replication Component
Both expression and cloning vectors contain a nucleic acid sequence
that enables the vector to replicate in one or more selected host
cells. Generally, in cloning vectors this sequence is one that
enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
Most expression vectors are "shuttle" vectors, i.e., they are
capable of replication in at least one class of organisms but can
be transfected into another organism for expression. For example, a
vector is cloned in E. coli and then the same vector is transfected
into yeast or mammalian cells for expression even though it is not
capable of replicating independently of the host cell
chromosome.
DNA may also be amplified by insertion into the host genome. This
is readily accomplished using Bacillus species as hosts, for
example, by including in the vector a DNA sequence that is
complementary to a sequence found in Bacillus genomic DNA.
Transfection of Bacillus with this vector results in homologous
recombination with the genome and insertion of the HRG2-.alpha.
DNA. However, the recovery of genomic DNA encoding the HRG2-.alpha.
is more complex than that of an exogenously replicated vector
because restriction enzyme digestion is required to excise the
HRG2-.alpha. DNA.
(iii) Selection Gene Component
Expression and cloning vectors should contain a selection gene,
also termed a selectable marker. This gene encodes a protein
necessary for the survival or growth of transformed host cells
grown in a selective culture medium. Host cells not transformed
with the vector containing the selection gene will not survive in
the culture medium. Typical selection genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media, e.g., the gene encoding D-alanine
racemase for Bacilli.
One example of a selection scheme utilizes a drug to arrest growth
of a host cell. Those cells that are successfully transformed with
a heterologous gene express a protein conferring drug resistance
and thus survive the selection regimen. Examples of such dominant
selection use the drugs neomycin (Southern et al., J. Molec. Appl.
Genet., 1: 327, 1982), mycophenolic acid (Mulligan et al., Science,
209: 1422, 1980) or hygromycin (Sugden et al., Mol. Cell. Biol., 5:
410-413, 1985). The three examples given above employ bacterial
genes under eukaryotic control to convey resistance to the
appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic
acid), or hygromycin, respectively.
Another example of suitable selectable markers for mammalian cells
are those that enable the identification of cells competent to take
up the HRG2-.alpha. nucleic acid, such as dihydrofolate reductase
(DHFR) or thymidine kinase. The mammalian cell transformants are
placed under selection pressure which only the transformants are
uniquely adapted to survive by virtue of having taken up the
marker. Selection pressure is imposed by culturing the
transformants under conditions in which the concentration of
selection agent in the medium is successively changed, thereby
leading to amplification of both the selection gene and the DNA
that encodes the HRG2-.alpha.. Amplification is the process by
which genes in greater demand for the production of a protein
critical for growth are reiterated in tandem within the chromosomes
of successive generations of recombinant cells. Increased
quantities of the HRG2-.alpha. are synthesized from the amplified
DNA.
For example, cells transformed with the DHFR selection gene are
first identified by culturing all of the transformants in a culture
medium that contains methotrexate (Mtx), a competitive antagonist
of DHFR. An appropriate host cell when wild-type DHFR is employed
is the Chinese hamster ovary (CHO) cell line deficient in DHFR
activity, prepared and propagated as described by Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216, 1980. The transformed
cells are then exposed to increased levels of methotrexate. This
leads to the synthesis of multiple copies of the DHFR gene, and,
concomitantly, multiple copies of other DNA comprising the
expression vectors, such as the DNA encoding the HRG2-.alpha.. This
amplification technique can be used with any otherwise suitable
host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of
endogenous DHFR if, for example, a mutant DHFR gene that is highly
resistant to Mtx is employed (EP 117,060). Alternatively, host
cells (particularly wild-type hosts that contain endogenous DHFR)
transformed or co-transformed with DNA sequences encoding the
HRG2-.alpha., wild-type DHFR protein, and another selectable marker
such as aminoglycoside 3' phosphotransferase (APH) can be selected
by cell growth in medium containing a selection agent for the
selectable marker such as an aminoglycosidic antibiotic, e.g.,
kanamycin, neomycin, or G418 (see U.S. Pat. No. 4,965,199).
A suitable selection gene for use in yeast is the trp1 gene present
in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39,
1979; Kingsman et al., Gene, 7: 141, 1979; or Tschemper et al.,
Gene, 10: 157, 1980). The trp1 gene provides a selection marker for
a mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12,
1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
(iv) Promoter Component
Expression and cloning vectors usually contain a promoter that is
recognized by the host organism and is operably linked to the
HRG2-.alpha. nucleic acid. Promoters are untranslated sequences
located upstream (5') to the start codon of a structural gene
(generally within about 100 to 1000 bp) that control the
transcription and translation of a particular nucleic acid
sequence, such as the HRG2-.alpha. to which they are operably
linked. Such promoters typically fall into two classes, inducible
and constitutive. Inducible promoters are promoters that initiate
increased levels of transcription from DNA under their control in
response to some change in culture conditions, e.g., the presence
or absence of a nutrient or a change in temperature. At this time a
large number of promoters recognized by a variety of potential host
cells are well known. These promoters are operably linked to DNA
encoding the HRG2-.alpha. by removing the promoter from the source
DNA by restriction enzyme digestion and inserting the isolated
promoter sequence into the vector. Both the native HRG2-.alpha.
promoter sequence and many heterologous promoters may be used to
direct amplification and/or expression of the HRG2-.alpha. DNA.
However, heterologous promoters are preferred, as they generally
permit greater transcription and higher yields of expressed
HRG2-.alpha. as compared to the native HRG2-.alpha. promoter.
Promoters suitable for use with prokaryotic hosts include the
.beta.-lactamase and lactose promoter systems (Chang et al.,
Nature, 275: 615, 1978; and Goeddel et al., Nature, 281: 544,
1979), alkaline phosphatase, a tryptophan (trp) promoter system
(Goeddel, Nucleic Acids Res., 8: 4057, 1980 and EP 36,776) and
hybrid promoters such as the tac promoter (deBoer et al., Proc.
Natl. Acad. Sci. USA, 80: 21-25, 1983). However, other known
bacterial promoters are suitable. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to DNA encoding the HRG2-.alpha. (Siebenlist et al.,
Cell, 20: 269, 1980) using linkers or adaptors to supply any
required restriction sites. Promoters for use in bacterial systems
also generally will contain a Shine-Dalgarno (S. D.) sequence
operably linked to the DNA encoding the HRG2-.beta..
Suitable promoting sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.
Chem., 255: 2073, 1980) or other glycolytic enzymes (Hess et al.,
J. Adv. Enzyme Reg., 7: 149, 1968; and Holland, Biochemistry, 17:
4900, 1978), such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the
additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in
Hitzeman et al., EP 73,657A. Yeast enhancers also are
advantageously used with yeast promoters.
Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CXCAAT (Seq. ID #1)region where X
may be any nucleotide. At the 3' end of most eukaryotic genes is an
AATAAA sequence (Seq. #2)that may be the signal for addition of the
poly A tail to the 3' end of the coding sequence. All of these
sequences are suitably inserted into mammalian expression
vectors.
HRG2-.alpha. transcription from vectors in mammalian host cells is
controlled by promoters obtained from the genomes of viruses such
as polyoma virus, fowlpox virus (UK 2,211,504, published 5 Jul.
1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B
virus and most preferably Simian Virus 40 (SV40), from heterologous
mammalian promoters, e.g., the actin promoter or an immunoglobulin
promoter, from heat-shock promoters, and from the promoter normally
associated with the HRG2-.alpha. sequence, provided such promoters
are compatible with the host cell systems.
The early and late promoters of the SV40 virus are conveniently
obtained as an SV40 restriction fragment that also contains the
SV40 viral origin of replication (Fiers et al., Nature, 273:113
(1978); Mulligan and Berg, Science, 209: 1422-1427 (1980); Pavlakis
et al., Proc. Natl. Acad. Sci. USA, 78: 7398-7402 (1981)). The
immediate early promoter of the human cytomegalovirus is
conveniently obtained as a HindIII E restriction fragment
(Greenaway et al., Gene, 18: 355-360 (1982)). A system for
expressing DNA in mammalian hosts using the bovine papilloma virus
as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification
of this system is described in U.S. Pat. No. 4,601,978. See also
Gray et al., Nature, 295: 503-508 (1982) on expressing cDNA
encoding immune interferon in monkey cells; Reyes et al., Nature,
297: 598-601 (1982) on expression of human .beta.-interferon cDNA
in mouse cells under the control of a thymidine kinase promoter
from herpes simplex virus; Canaani and Berg, Proc. Natl. Acad. Sci.
USA, 79: 5166-5170 (1982) on expression of the human interferon
.beta.1gene in cultured mouse and rabbit cells; and Gorman et al.,
Proc. Natl. Acad. Sci. USA, 79: 6777-6781 (1982) on expression of
bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo
fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse
NIH-3T3 cells using the Rous sarcoma virus long terminal repeat as
a promoter.
(v) Enhancer Element Component
Transcription of a DNA encoding the HRG2-.alpha. of this invention
by higher eukaryotes is often increased by inserting an enhancer
sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10-300 bp, that act on a promoter to increase
its transcription. Enhancers are relatively orientation and
position independent having been found 5' (Laimins et al., Proc.
Natl. Acad. Sci. USA, 78: 993, 1981) and 3' (Lusky et al., Mol.
Cell Bio., 3: 1108, 1983) to the transcription unit, within an
intron (Banerji et al., Cell, 33: 729, 1983) as well as within the
coding sequence itself (Osborne et al., Mol. Cell Bio., 4: 1293,
1984). Many enhancer sequences are now known from mammalian genes
(globin, elastase, albumin, .alpha.-fetoprotein and insulin).
Typically, however, one will use an enhancer from a eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers (see also Yaniv, Nature, 297:
17-18 (1982)) on enhancing elements for activation of eukaryotic
promoters. The enhancer may be spliced into the vector at a
position 5' or 3' to the HRG2-.alpha. DNA, but is preferably
located at a site 5' from the promoter.
(vi) Transcription Termination Component
Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3'
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding the
HRG2-.alpha.. The 3' untranslated regions also include
transcription termination sites.
Construction of suitable vectors containing one or more of the
above listed components the desired coding and control sequences
employs standard ligation techniques. Isolated plasmids or DNA
fragments are cleaved, tailored, and religated in the form desired
to generate the plasmids required.
For analysis to confirm correct sequences in plasmids constructed,
the ligation mixtures are used to transform E. coli K12 strain 294
(ATCC 31,446) and successful transformants selected by ampicillin
or tetracycline resistance where appropriate. Plasmids from the
transformants are prepared, analyzed by restriction endonuclease
digestion, and/or sequenced by the method of Messing et al.,
Nucleic Acids Res., 9: 309 (1981) or by the method of Maxam et al.,
Methods in Enzymology, 65: 499 (1980).
Particularly useful in the practice of this invention are
expression vectors that provide for the transient expression in
mammalian cells of DNA encoding the HRG2-.alpha.. In general,
transient expression involves the use of an expression vector that
is able to replicate efficiently in a host cell, such that the host
cell accumulates many copies of the expression vector and, in turn,
synthesizes high levels of a desired polypeptide encoded by the
expression vector. Transient expression systems, comprising a
suitable expression vector and a host cell, allow for the
convenient positive identification of polypeptides encoded by
cloned DNAs, as well as for the rapid screening of such
polypeptides for desired biological or physiological properties.
Thus, transient expression systems are particularly useful in the
invention for purposes of identifying analogs and variants of the
HRG2-.alpha. that have HRG2-like activity. Such a transient
expression system is described in patent application U.S. Ser. No.
07/101,712 now U.S. Pat. No. 5,024,939, issued Jun. 18, 1991.
Other methods, vectors, and host cells suitable for adaptation to
the synthesis of the HRG2-.alpha. in recombinant vertebrate cell
culture are described in Gething et al., Nature, 293: 620-625,
1981; Mantei et al., Nature, 281: 40-46, 1979; Levinson et al.; EP
117,060; and EP 117,058. A particularly useful expression plasmid
for mammalian cell culture expression of the HRG2-.alpha. is pRK5
(EP pub. no.307,24 4) or pSVI6B (U.S. Ser. No. 07/441,574, filed 22
Nov. 1989, abandoned Apr. 4, 1991 the disclosure of which is
incorporated herein by reference).
D. Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the vectors herein
are the prokaryote, yeast, or higher eukaryote cells described
above. Suitable prokaryotes include eubacteria, such as
Gram-negative or Gram-positive organisms, for example, E. coli ,
Bacilli such as B. subtilis, Pseudomonas species such as P.
aeruginosa, Salmonella typhimurium, or Serratia marcescans. One
preferred E. coli cloning host is E. coli 294 (ATCC 31,446),
although other strains such as E. coli B, E. coli .sub.x 1776 (ATCC
31,537), and E. coli W3110 (ATCC 27,325) are suitable. These
examples are illustrative rather than limiting. Preferably the host
cell should secrete minimal amounts of proteolytic enzymes.
Alternatively, in vitro methods of cloning, e.g., PCR or other
nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are suitable hosts for HRG2-encoding vectors.
Saccharomyces cerevisiae, or common baker's yeast, is the most
commonly used among lower eukaryotic host microorganisms. However,
a number of other genera, species, and strains are commonly
available and useful herein, such as Schizosaccharomyces pombe
(Beach and Nurse, Nature, 290: 140 (1981); EP 139,383, published
May 2, 1985), Kluyveromyces hosts (U.S. Pat. No. 4,943,529) such
as, e.g., K. lactis (Louvencourt et al., J. Bacteriol., 737 (1983);
K. fragilis, K. bulgaricus, K. thermotolerans, and K. marxianus,
yarrowia (EP 402,226); Pichia pastoris (EP 183,070), Sreekrishna et
al., J. Basic Microbiol., 28: 265-278 (1988); Candida, Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76: 5259-5263 (1979), and filamentous fungi such
as, e.g, Neurospora, Penicillium, Tolypocladium (WO 91/00357,
published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans
(Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289
(1983); Tilburn et al., Gene, 26: 205-221 (1983); Yelton et al.,
Proc. Natl. Acad. Sci. USA, 81: 1470-1474 (1984) and A. niger
(Kelly and Hynes, EMBO J., 4: 475-479 (1985)).
Suitable host cells for the expression of glycosylated HRG2-.alpha.
polypeptide are derived from multicellular organisms. Such host
cells are capable of complex processing and glycosylation
activities. In principle, any higher eukaryotic cell culture is
workable, whether from vertebrate or invertebrate culture. Examples
of invertebrate cells include plant and insect cells. Numerous
baculoviral strains and variants and corresponding permissive
insect host cells from hosts such as Spodoptera frugiperda
(caterpillar), Aedes aegypti (mosquito), Aedes albopictus
(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
host cells have been identified (see, e.g., Luckow et al.,
Bio/Technology, 6: 47-55 (1988); Miller et al., in Genetic
Engineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,
1986), pp. 277-279; and Maeda et al., Nature, 315: 592-594 (1985)).
A variety of such viral strains are publicly available, e.g., the
L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein
according to the present invention, particularly for transfection
of Spodoptera frugiperda cells. Plant cell cultures of cotton,
corn, potato, soybean, petunia, tomato, and tobacco can be utilized
as hosts. Typically, plant cells are transfected by incubation with
certain strains of the bacterium Agrobacterium tumefaciens, which
has been previously manipulated to contain the HRG2-.alpha. DNA.
During incubation of the plant cell culture with A. tumefaciens,
the DNA encoding HRG2 is transferred to the plant cell host such
that it is transfected, and will, under appropriate conditions,
express the HRG2-.alpha. DNA. In addition, regulatory and signal
sequences compatible with plant cells are available, such as the
nopaline synthase promoter and polyadenylation signal sequences
(Depicker et al., J. Mol. Appl. Gen., 1: 561 (1982)). In addition,
DNA segments isolated from the upstream region of the T-DNA 780
gene are capable of activating or increasing transcription levels
of plant-expressible genes in recombinant DNA-containing plant
tissue (see EP 321,196, published 21 Jun. 1989).
However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure in recent years (Tissue Culture,
Academic Press, Kruse and Patterson, editors (1973)). Examples of
useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol., 36: 59, 1977); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 [1980]);
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251
[1980]); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells
(Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 [1982]); MRC 5
cells; FS4 cells; and a human hepatoma cell line (Hep G2).
Preferred host cells are human embryonic kidney 293 and Chinese
hamster ovary cells.
Host cells are transfected and preferably transformed with the
above-described expression or cloning vectors of this invention and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
Transfection refers to the taking up of an expression vector by a
host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO.sub.4 and
electroporation. Successful transfection is generally recognized
when any indication of the operation of this vector occurs within
the host cell.
Transformation means introducing DNA into an organism so that the
DNA is replicable, either as an extrachromosomal element or by
chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as
described in section 1.82 of Sambrook et al., supra, is generally
used for prokaryotes or other cells that contain substantial
cell-wall barriers. Infection with Agrobacterium tumefaciens is
used for transformation of certain plant cells, as described by
Shaw et al., Gene, 23: 315 (1983) and WO 89/05859, published 29
Jun. 1989. For mammalian cells without such cell walls, the calcium
phosphate precipitation method described in sections 16.30-16.37 of
Sambrook et al, supra, is preferred. General aspects of mammalian
cell host system transformations have been described by Axel in
U.S. Pat. No. 4,399,216, issued 16 Aug. 1983. Transformations into
yeast are typically carried out according to the method of Van
Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc.
Natl. Acad. Sci. (USA), 76: 3829 (1979). However, other methods for
introducing DNA into cells such as by nuclear injection,
electroporation, or protoplast fusion may also be used.
E. Culturing the Host Cells
Prokaryotic cells used to produce the HRG2-.alpha. polypeptide of
this invention are cultured in suitable media as described
generally in Sambrook et al., supra.
The mammalian host cells used to produce the HRG2-.alpha. of this
invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ([DMEM], Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham and Wallace, Meth.
Enz., 58: 44 (1979), Barnes and Sato, Anal. Biochem., 102: 255
(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or
4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985; or
copending U.S. Ser. No. 07/592,107 U.S. Pat. No. 5,122,469 or U.S.
Pat. No. 07/592,141, abandoned, both filed on 3 Oct. 1990, the
disclosures of all of which are incorporated herein by reference,
may be used as culture media for the host cells. Any of these media
may be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor),
salts (such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and
thymidine), antibiotics (such as Gentamycin.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
The host cells referred to in this disclosure encompass cells in in
vitro culture as well as cells that are within a host animal.
It is further envisioned that the HRG2-.alpha. of this invention
may be produced by homologous recombination, or with recombinant
production methods utilizing control elements introduced into cells
already containing DNA encoding the HRG2-.alpha. currently in use
in the field. For example, a powerful promoter/enhancer element, a
suppressor, or an exogenous transcription modulatory element is
inserted in the genome of the intended host cell in proximity and
orientation sufficient to influence the transcription of DNA
encoding the desired HRG2-.alpha.. The control element does not
encode the HRG2 of this invention, but the DNA is present in the
host cell genome. One next screens for cells making the
HRG2-.alpha. of this invention, or increased or decreased levels of
expression, as desired.
F. Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample
directly, for example, by conventional Southern blotting, Northern
blotting to quantitate the transcription of mRNA (Thomas, Proc.
Natl. Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe based on the sequences provided herein. Various labels may be
employed, most commonly radioisotopes, particularly .sup.32 P.
However, other techniques may also be employed, such as using
biotin-modified nucleotides for introduction into a polynucleotide.
The biotin then serves as the site for binding to avidin or
antibodies which may be labeled with a wide variety of labels, such
as radionuclides, fluorescers, enzymes, or the like.
Alternatively, antibodies may be employed that can recognize
specific duplexes, including DNA duplexes, RNA duplexes, and
DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in
turn may be labeled and the assay may be carried out where the
duplex is bound to a surface, so that upon the formation of duplex
on the surface, the presence of antibody bound to the duplex can be
detected.
Gene expression, alternatively, may be measured by immunological
methods, such as immunohistochemical staining of tissue sections
and assay of cell culture or body fluids, to quantitate directly
the expression of gene product. With immunohistochemical staining
techniques, a cell sample is prepared, typically by dehydration and
fixation, followed by reaction with labeled antibodies specific for
the gene product coupled where the labels are usually visually
detectable such as enzymatic labels, fluorescent labels,
luminescent labels, and the like. A particularly sensitive staining
technique suitable for use in the present invention is described by
Hsu et al., Am. J. Clin. Path., 75: 734-738 (1980).
Antibodies useful for immunohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be
prepared in any mammal. Conveniently, the antibodies may be
prepared against a native HRG2-.alpha. polypeptide or against a
synthetic peptide based on the DNA sequences provided herein as
described further in Section 4 below.
G. Purification of The Hereeulin 2-.alpha. Polypeptide
The HRG2-.alpha. may be recovered from a cellular membrane
fraction. Alternatively, a proteolyticly cleaved on a truncated
expressed soluble HRG2-.alpha. ligand may be recovered from the
culture medium as a soluble polypeptide. A HRG2-.alpha. may also be
recovered from host cell lysates when directly expressed without a
secretory signal.
When the HRG2-.alpha. is expressed in a recombinant cell other than
one of human origin, the HRG2-.alpha. is completely free of
proteins or polypeptides of human origin. However, it is necessary
to purify the HRG2-.alpha. from recombinant cell proteins or
polypeptides to obtain preparations that are substantially
homogeneous as to the HRG2-.alpha.. As a first step, the culture
medium or lysate is centrifuged to remove particulate cell debris.
The membrane and soluble protein fractions are then separated. The
HRG2-.alpha. may then be purified from both the soluble protein
fraction (requiring the presence of a protease) and from the
membrane fraction of the culture lysate, depending on whether the
HRG2-.alpha. is membrane bound. The following procedures are
exemplary of suitable purification procedures: fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reverse phase HPLC; chromatography on silica, heparin sepharose or
on a cation exchange resin such as DEAE; chromatofocusing;
SDS-PAGE; ammonium sulfate precipitation; and gel filtration using,
for example, Sephadex G-75.
HRG2-.alpha. variants in which residues have been deleted, inserted
or substituted are recovered in the same fashion as the native
HRG2-.alpha., taking account of any substantial changes in
properties occasioned by the variation. For example, preparation of
a HRG2-.alpha. fusion with another protein or polypeptide, e.g., a
bacterial or viral antigen, facilitates purification; an
immunoaffinity column containing antibody to the antigen can be
used to adsorb the fusion. Immunoaffinity columns such as a rabbit
polyclonal anti-HRG2 column can be employed to absorb the HRG2
variant by binding it to at least one remaining immune epitope. A
protease inhibitor such as phenylmethylsulfonylfluoride (PMSF) also
may be useful to inhibit proteolytic degradation during
purification, and antibiotics may be included to prevent the growth
of adventitious contaminants. One skilled in the art will
appreciate that purification methods suitable for native
HRG2-.alpha. may require modification to account for changes in the
character of the HRG2-.alpha. or its variants upon expression in
recombinant cell culture.
H. Covalent Modifications of HRG2-.alpha. Polypeptides
Covalent modifications of HRG2-.alpha. polypeptides are included
within the scope of this invention. Both native HRG2-.alpha. and
amino acid sequence variants of the HRG2-.alpha. may be covalently
modified. One type of covalent modification included within the
scope of this invention is a HRG2-.alpha. polypeptide fragment.
HRG2-.alpha. fragments having up to about 40 amino acid residues
may be conveniently prepared by chemical synthesis, or by enzymatic
or chemical cleavage of the full-length HRG2-.alpha. polypeptide or
HRG2-.alpha. variant polypeptide. Other types of covalent
modifications of the HRG2-.alpha. or fragments thereof are
introduced into the molecule by reacting targeted amino acid
residues of the HRG2-.alpha. or fragments thereof with an organic
derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues.
Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Para-bromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1M sodium
cacodylate at pH 6.0.
Lysinyl and amino terminal residues are reacted with succinic or
other carboxylic acid anhydrides. Derivatization with these agents
has the effect of reversing the charge of the lysinyl residues.
Other suitable reagents for derivatizing .alpha.-amino-containing
residues include imidoesters such as methyl picolinimidate;
pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione;
and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several
conventional reagents, among them phenylglyoxal, 2,3-butanedione,
1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine
residues requires that the reaction be performed in alkaline
conditions because of the high pK.sub.a of the guanidine functional
group. Furthermore, these reagents may react with the groups of
lysine as well as the arginine epsilon-amino group.
The specific modification of tyrosyl residues may be made, with
particular interest in introducing spectral labels into tyrosyl
residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using .sup.125 I or .sup.131 I to prepare labeled proteins for use
in radioimmunoassay, the chloramine T method described above being
suitable.
Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R'--N.dbd.C.dbd.N--R'),
where R and R' are different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
Derivatization with bifunctional agents is useful for crosslinking
HRG2-.alpha. to a water-insoluble support matrix or surface for use
in the method for purifying anti-HRG2-.alpha. antibodies, and vice
versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 are employed for protein immobilization.
Glutaminyl and asparaginyl residues are frequently deamidated to
the corresponding glutamyl and aspartyl residues, respectively.
Alternatively, these residues are deamidated under mildly acidic
conditions. Either form of these residues falls within the scope of
this invention.
Other modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
79-86 [1983]), acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
Another type of modification of the HRG-.alpha. polypeptide is the
formation of fusion proteins with a heterologous polypeptide. The
heterologous polypeptide may be an anchor sequence such as that
found in the decay accelerating system (DAF). The heterologous
polypeptide may be a toxin such as ricin, pseudomonas exotoxin,
gelonin, or other polypeptide that will result in target cell
death. Still other proteins may be fused to the HRG-.alpha.
polypeptide such as enzymes that result in cell death or inhibition
such as nucleases, including both DNAse and RNAse. These
heterheterologous polypeptides may alternatively be covalently
coupled to the HRG-.alpha. polypeptide. Similarly, other molecules
toxic or inhibitory to a target mammalian cell may be coupled to
the HRG-.alpha. polypeptide, such as antisense DNA that blocks gene
function or expression, and tricothecenes.
Another type of covalent modification of the HRG2-.alpha.
polypeptide included within the scope of this invention comprises
altering the native glycosylation pattern of the polypeptide. By
altering is meant deleting one or more carbohydrate moieties found
in native HRG2-.alpha., and/or adding one or more glycosylation
sites that are not present in the native HRG2-.alpha.
polypeptide.
Glycosylation of polypeptides is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tri-peptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tri-peptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose, to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the HRG2-.alpha. polypeptide is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tri-peptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the native HRG2-.alpha. sequence
(for O-linked glycosylation sites). For ease, the HRG2-.alpha.
amino acid sequence is preferably altered through changes at the
DNA level, particularly by mutating the DNA encoding the
HRG2-.alpha. polypeptide at preselected bases such that codons are
generated that will translate into the desired amino acids. The DNA
mutation(s) may be made using methods described above under the
heading of "Amino Acid Sequence Variants of HRG2-.alpha.
Polypeptide".
Another means of increasing the number of carbohydrate moieties on
the HRG2-.alpha. polypeptide is by chemical or enzymatic coupling
of glycosides to the polypeptide. These procedures are advantageous
in that they do not require production of the polypeptide in a host
cell that has glycosylation capabilities for N- and O-linked
glycosylation. Depending on the coupling mode used, the sugar(s)
may be attached to (a) arginine and histidine, (b) free carboxyl
groups, (c) free sulfhydryl groups such as those of cysteine, (d)
free hydroxyl groups such as those of serine, threonine, or
hydroxyproline, (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan, or (f) the amide group of
glutamine. These methods are described in WO 87/05330, published 11
Sep. 1987, and in Aplin and Wriston (CRC Crit. Rev. Biochem., pp.
259-306 [1981]).
Removal of carbohydrate moieties present on the native HRG2-.alpha.
polypeptide may be accomplished chemically or enzymatically.
Chemical deglycosylation requires exposure of the polypeptide to
the compound trifluoromethanesulfonic acid, or an equivalent
compound. This treatment results in the cleavage of most or all
sugars except the linking sugar (N-acetylglucosamine or
N-acetylgalactosamine), while leaving the polypeptide intact.
Chemical deglycosylation is described by Hakimuddin et al. (Arch.
Biochem. Biophys., 259:52 [1987]) and by Edge et al. (Anal.
Biochem., 118:131 [1981]). Enzymatic cleavage of carbohydrate
moieties on polypeptides can be achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et al. (Meth.
Enzymol., 138:350 [1987]).
Glycosylation at potential glycosylation sites may be prevented by
the use of the compound tunicamycin as described by Duskin et al.
(J. Biol. Chem., 257:3105 [1982]). Tunicamycin blocks the formation
of protein-N-glycoside linkages.
Another type of covalent modification of the HRG2 comprises linking
the HRG2-.alpha. polypeptide to various nonproteinaceous polymers,
e.g., polyethylene glycol, polypropylene glycol or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
One preferred way to improve the in vivo circulating half life of
HRG2-.alpha. is to conjugate it to a polymer that confers extended
half-life, such as conjugating polyethylene glycol (PEG) to HRG2,
was found to be an excellent way to increase the half-life. PEG is
an non-immunogenic, linear, uncharged polymer with three water
molecules per ethylene oxide unit which therefore can alter the
hydrodynamic properties of the conjugated molecules dramatically.
(Maxfield, et al, Polymer 16,505-509 [1975]; Bailey, F. E., et al,
in Nonionic Surfactants [Schick, M. J., ed]pp.794-821, 1967).
Several enzymes for therapeutic usage were PEGylated to increase
the in vivo half-life effectively (Abuchowski, A. et al, J. Biol.
Chem. 252, 3582-3586, 1977; Abuchowski, A. et al, Cancer Biochem.
Biophys. 7, 175-186, 1984). PEGylation of IL-2(interleukin-2) was
also reported to increase circulatory life as well as its potency
(Katre, N. V. et al, Proc. Natl. Acad. Sci., 84, 1487-1491, 1987;
Goodson, R. et al Bio/Technology, 8, 343-346, 1990). PEGylation of
other molecules were reported to have reduced immunogenicity and
toxicity (Abuchowski, A. et al, J. Biol. Chem., 252, 3578-3581,
1977).
The HRG2-.alpha. also may be entrapped in microcapsules prepared,
for example, by coacervation techniques or by interfacial
polymerization (for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-[methylmethacylate] microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.,
Ed., (1980).
HRG2-.alpha. preparations are also useful in generating antibodies,
as standards in assays for the HRG2-.alpha. (e.g., by labeling the
HRG2 for use as a standard in a radioimmunoassay, enzyme-linked
immunoassay, or radioreceptor assay), in affinity purification
techniques, and in competitive-type receptor binding assays when
labeled with radioiodine, enzymes, fluorophores, spin labels, and
the like.
Since it is often difficult to predict in advance the
characteristics of a variant HRG2-.alpha. , it will be appreciated
that some screening of the recovered variant will be needed to
select the optimal variant. For example, a change in the
immunological character of the HRG2-.alpha. molecule, such as
affinity for a given antibody, is measured by a competitive-type
immunoassay. The variant is assayed for binding affinity to
HER2-.alpha. or to other receptors. The variant is assayed for
changes in the suppression or enhancement of its activity by
comparison to the activity observed for native HRG2-.alpha. in the
same assay. Other potential modifications of protein or polypeptide
properties such as redox or thermal stability, hydrophobicity,
susceptibility to proteolytic degradation, stability in recombinant
cell culture or in plasma, or the tendency to aggregate with
carriers or into multimers are assayed by methods well known in the
art.
2. Therapeutic Compositions and Administration of HRG2-.alpha.
Therapeutic formulations of HRG2 or HRG2-antibody are prepared for
storage by mixing HRG2 having the desired degree of purity with
optional physiologically acceptable carriers, excipients, or
stabilizers (Remington's Pharmaceutical Sciences, supra), in the
form of lyophilized cake or aqueous solutions. Acceptable carriers,
excipients or stabilizers are nontoxic to recipients at the dosages
and concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
Tween, Pluronics or polyethylene glycol (PEG).
The HRG2 or HRG2-antibody to be used for in vivo administration
must be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to or following lyophilization
and reconstitution. The HRG2 or its antibody ordinarily will be
stored in lyophilized form or in solution.
Therapeutic HRG2, its antibody compositions generally are placed
into a container having a sterile access port, for example, an
intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
The HRG2, its antibody or HRG2 variant when used as an antagonist
may be optionally combined with or administered in concert with
other agents known for use in the treatment of particular malignant
or cancerous disorders. When HRG2 is used as an agonist to
stimulate the HER2 receptor, it may be combined with or
administered in concert with other compositions that stimulate
growth.
The route of HRG2-.alpha. or HRG2-.alpha. antibody administration
is in accord with known methods, e.g., injection or infusion by
intravenous, intraperitoneal, intracerebral, intramuscular,
intraocular, intraarterial, or intralesional routes, or by
sustained release systems as noted below. The HRG2-.alpha. is
administered continuously by infusion or by bolus injection.
HRG2-.alpha. antibody is administered in the same fashion, or by
administration into the blood stream or lymph.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
protein, which matrices are in the form of shaped articles, e.g.
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels [e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.
Biomed. Mater. Res., 15: 167-277 [1981] and Langer, Chem. Tech.,
12: 98-105 [1982] or poly(vinylalcohol)], polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 [1983]),
non-degradable ethylene-vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the Lupron
Depot.TM. (injectable micropheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). While polymers such
as ethylene-vinyl acetate and lactic acid-glycolic acid enable
release of molecules for over 100 days, certain hydrogels release
proteins for shorter time periods. When encapsulated proteins
remain in the body for a long time, they may denature or aggregate
as a result of exposure to moisutre at 37.degree. C., resulting in
a loss of biological activity and possible changes in
immunogenicity. Rational strategies can be devised for protein
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism is discovered to be intermolecular S--S
bond formation through thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
Sustained-release HRG2 or antibody compositions also include
liposomally entrapped HRG2 or antibody. Liposomes containing HRG2
or antibody are prepared by methods known per se: DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985);
Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP
52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese
patent application 83-118008; U.S. Pat. Nos. 4,485,045 and
4,544,545; and EP 102,324. Ordinarily the liposomes are of the
small (about 200-800 Angstroms) unilamelar type in which the lipid
content is greater than about 30 mol. % cholesterol, the selected
proportion being adjusted for the optimal HRG2 therapy. Liposomes
with enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
Another use of the present invention comprises incorporating HRG2
polypeptide or antibody into formed articles. Such articles can be
used in modulating cellular growth and development. In addition,
cell growth and division and tumor invasion may be modulated with
these articles.
An effective amount of HRG2 or antibody to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
patient. Accordingly, it will be necessary for the therapist to
titer the dosage and modify the route of administration as required
to obtain the optimal therapeutic effect. A typical daily dosage
might range from about 1 .mu.g/kg to up to 100 mg/kg or more,
depending on the factors mentioned above. Typically, the clinician
will administer the HRG2 or antibody until a dosage is reached that
achieves the desired effect. The progress of this therapy is easily
monitored by conventional assays.
3. Heregulin 2-.alpha. Antibody Preparation and Therapeutic Use
The antibodies of this invention are obtained by routine screening.
Polyclonal antibodies to the HRG2-.alpha. generally are raised in
animals by multiple subcutaneous (sc) or intraperitoneal (ip)
injections of the HRG2-.alpha. and an adjuvant. It may be useful to
conjugate the HRG2-.alpha. or a fragment containing the target
amino acid sequence to a protein that is immunogenic in the species
to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,
bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1 N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
The route and schedule of the host animal or cultured
antibody-producing cells therefrom are generally in keeping with
established and conventional techniques for antibody stimulation
and production. While mice are frequently employed as the test
model, it is contemplated that any mammalian subject including
human subjects or antibody-producing cells obtained therefrom can
be manipulated according to the processes of this invention to
serve as the basis for production of mammalian, including human,
hybrid cell lines.
Animals are typically immunized against the immunogenic conjugates
or derivatives by combining 1 mg or 1 .mu.g of conjugate (for
rabbits or mice, respectively) with 3 volumes of Freund's complete
adjuvant and injecting the solution intradermally at multiple
sites. One month later the animals are boosted with 1/5 to 1/10 the
original amount of conjugate in Freund's complete adjuvant (or
other suitable adjuvant) by subcutaneous injection at multiple
sites. 7 to 14 days later animals are bled and the serum is assayed
for anti-HRG2-.alpha. antibody titer. Animals are boosted until the
titer plateaus. Preferably, the animal is boosted with the
conjugate of the same HRG2-.alpha. , but conjugated to a different
protein and/or through a different cross-linking agent. Conjugates
also can be made in recombinant cell culture as protein fusions.
Also, aggregating agents such as alum are used to enhance the
immune response.
After immunization, monoclonal antibodies are prepared by
recovering immune lymphoid cells--typically spleen cells or
lymphocytes from lymph node tissue--from immunized animals and
immortalizing the cells in conventional fashion, e.g., by fusion
with myeloma cells or by Epstein-Barr (EB)-virus transformation and
screening for clones expressing the desired antibody. The hybridoma
technique described originally by Kohler and Milstein, Eur. J.
Immunol. 6:511 (1976) has been widely applied to produce hybrid
cell lines that secrete high levels of monoclonal antibodies
against many specific antigens.
It is possible to fuse cells of one species with another. However,
it is preferable that the source of the immunized antibody
producing cells and the myeloma be from the same species.
The hybrid cell lines can be maintained in culture in vitro in cell
culture media. The cell lines of this invention can be selected
and/or maintained in a composition comprising the continuous cell
line in hypoxanthine-aminopterin thymidine (HAT) medium. In fact,
once the hybridoma cell line is established, it can be maintained
on a variety of nutritionally adequate media. Moreover, the hybrid
cell lines can be stored and preserved in any number of
conventional ways, including freezing and storage under liquid
nitrogen. Frozen cell lines can be revived and cultured
indefinitely with resumed synthesis and secretion of monoclonal
antibody. The secreted antibody is recovered from tissue culture
supernatant by conventional methods such as precipitation, ion
exchange chromatography, affinity chromatography, or the like. The
antibodies described herein are also recovered from hybridoma cell
cultures by conventional methods for purification of IgG or IgM as
the case may be that heretofore have been used to purify these
immunoglobulins from pooled plasma, e.g., ethanol or polyethylene
glycol precipitation procedures. The purified antibodies are
sterile filtered, and optionally are conjugated to a detectable
marker such as an enzyme or spin label for use in diagnostic assays
of the HRG2 in test samples.
While routinely mouse monoclonal antibodies are used, the invention
is not so limited; in fact, human antibodies may be used and may
prove to be preferable. Such antibodies can be obtained by using
human hybridomas (Cote et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985)). In fact, according to the
invention, techniques developed for the production of chimeric
antibodies (Morrison et al., Proc. Natl. Acad. Sci., 81:6851
(1984); Neuberger et al., Nature 312:604 (1984); Takeda et al.,
Nature 314:452 (1985)) by splicing the genes from a mouse antibody
molecule of appropriate antigen specificity together with genes
from a human antibody molecule of appropriate biological activity
(such as ability to activate human complement and mediate ADCC) can
be used; such antibodies are within the scope of this invention.
Techniques for creating recombinant DNA versions of the
antigen-binding regions of antibody molecules (known as Fab or
variable regions fragments) which bypass the generation of
monoclonal antibodies are encompassed within the practice of this
invention. One extracts antibody-specific messenger RNA molecules
from immune system cells taken from an immunized animal,
transcribes these into complementary DNA (cDNA), and clones the
cDNA into a bacterial expression system. One example of such a
technique suitable for the practice of this invention was developed
by researchers at Scripps/Stratagene, and incorporates a
bacteriophage lambda vector system which contains a leader sequence
that causes the expressed Fab protein to migrate to the periplasmic
space (between the bacterial cell membrane and the cell wall) or to
be secreted.
One can rapidly generate and screen great numbers of functional Fab
fragments for those which bind the antigen. Such HRG2-binding
molecules (Fab fragments with specificity for the HRG2) are
specifically encompassed within the term "antibody" as it is
defined, discussed, and claimed herein.
The HRG2-.alpha. and HRG2-.beta. antibodies of this invention
preferably do not cross-react with other members of the EGF family
(FIG. 6) or with each other.
The antibodies of this invention are also useful in passively
immunizing patients.
4. Non-Therapeutic Uses of Heregulin 2-.alpha. and its
Antibodies
The nucleic acid encoding the HRG2-.alpha. may be used as a
diagnostic for tissue specific typing. For example, such procedures
as in situ hybridization, and Northern and Southern blotting, and
PCR analysis may be used to determine whether DNA and/or RNA
encoding the HRG2-.alpha. are present in the cell type(s) being
evaluated. In particular, the nucleic acid may be useful as a
specific probe for certain types of tumor cells such as, for
example, mammary gland, gastric and colon adenocarcinomas, salivary
gland and other tissues containing the p185.sup.HER2.
Isolated HRG2-.alpha. polypeptide may be used in quantitative
diagnostic assays as a standard or control against which samples
containing unknown quantities of HRG2-.alpha. may be compared.
HRG2-.alpha. antibodies are useful in diagnostic assays for HRG2
expression in specific cells or tissues. The antibodies are labeled
in the same fashion as the HRG2-.alpha. described above and/or are
immobilized on an insoluble matrix.
HRG2-.alpha. antibodies also are useful for the affinity
purification of the HRG2-.alpha. from recombinant cell culture or
natural sources. The HRG2-.alpha. antibodies that do not detectably
cross-react with other HRG2-.alpha. can be used to purify
HRG2-.alpha. free from other known ligands or contaminating
protein.
Suitable diagnostic assays for the HRG2-.alpha. and its antibodies
are well known per se. Such assays include competitive and sandwich
assays, and steric inhibition assays. Competitive and sandwich
methods employ a phase-separation step as an integral part of the
method while steric inhibition assays are conducted in a single
reaction mixture. Fundamentally, the same procedures are used for
the assay of the HRG2-.alpha. and for substances that bind the
HRG2-.alpha. , although certain methods will be favored depending
upon the molecular weight of the substance being assayed.
Therefore, the substance to be tested is referred to herein as an
analyte, irrespective of its status otherwise as an antigen or
antibody, and proteins that bind to the analyte are denominated
binding partners, whether they be antibodies, cell surface
receptors, or antigens.
Analytical methods for the HRG2-.alpha. or its anitbodies all use
one or more of the following reagents: labeled analyte analogue,
immobilized analyte analogue, labeled binding partner, immobilized
binding partner and steric conjugates. The labeled reagents also
are known as "tracers."
The label used (and this is also useful to label HRG2-.alpha.
encoding nucleic acid for use as a probe) is any detectable
functionality that does not interfere with the binding of analyte
and its binding partner. Numerous labels are known for use in
immunoassay, examples including moieties that may be detected
directly, such as fluorochrome, chemiluminescent, and radioactive
labels, as well as moieties, such as enzymes, that must be reacted
or derivatized to be detected. Examples of such labels include the
radioisotopes .sup.32 P, .sup.14 C, .sup.125 I, .sup.3 H, and
.sup.1311 I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luciferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
Conventional methods are available to bind these labels covalently
to proteins or polypeptides. For instance, coupling agents such as
dialdehydes, carbodiimides, dimaleimides, bis-imidates,
bis-diazotized benzidine, and the like may be used to tag the
antibodies with the above-described fluorescent, chemiluminescent,
and enzyme labels. See, for example, U.S. Pat. No. 3,940,475
(fluorimetry) and U.S. Pat. No. 3,645,090 (enzymes); Hunter et al.,
Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014-1021
(1974); Pain et al., J. Immunol. Methods, 40: 219-230 (1981); and
Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982). Preferred
labels herein are enzymes such as horseradish peroxidase and
alkaline phosphatase. The conjugation of such label, including the
enzymes, to the antibody is a standard manipulative procedure for
one of ordinary skill in immunoassay techniques. See, for example,
O'Sullivan et al., "Methods for the Preparation of Enzyme-antibody
Conjugates for Use in Enzyme Immunoassay," in Methods in
Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic
Press, New York, N.Y., 1981), pp. 147-166. Such bonding methods are
suitable for use with HRG2-.alpha. or its antibodies, all of which
are proteinaceous.
Immobilization of reagents is required for certain assay methods.
Immobilization entails separating the binding partner from any
analyte that remains free in solution. This conventionally is
accomplished by either insolubilizing the binding partner or
analyte analogue before the assay procedure, as by adsorption to a
water-insoluble matrix or surface (Bennich et al., U.S. Pat. No.
3,720,760), by covalent coupling (for example, using glutaraldehyde
cross-linking), or by insolubilizing the partner or analogue
afterward, e.g., by immunoprecipitation.
Other assay methods, known as competitive or sandwich assays, are
well established and widely used in the commercial diagnostics
industry.
Competitive assays rely on the ability of a tracer analogue to
compete with the test sample analyte for a limited number of
binding sites on a common binding partner. The binding partner
generally is insolubilized before or after the competition and then
the tracer and analyte bound to the binding partner are separated
from the unbound tracer and analyte. This separation is
accomplished by decanting (where the binding partner was
preinsolubilized) or by centrifuging (where the binding partner was
precipitated after the competitive reaction). The amount of test
sample analyte is inversely proportional to the amount of bound
tracer as measured by the amount of marker substance. Dose-response
curves with known amounts of analyte are prepared and compared with
the test results to quantitatively determine the amount of analyte
present in the test sample. These assays are called ELISA systems
when enzymes are used as the detectable markers.
Another species of competitive assay, called a "homogeneous" assay,
does not require a phase separation. Here, a conjugate of an enzyme
with the analyte is prepared and used such that when anti-analyte
binds to the analyte the presence of the anti-analyte modifies the
enzyme activity. In this case, the HRG2-.alpha. or its
immunologically active fragments are conjugated with a bifunctional
organic bridge to an enzyme such as peroxidase. Conjugates are
selected for use with anti-HRG2-.alpha. so that binding of the
anti-HRG2-.alpha. antibody inhibits or potentiates the enzyme
activity of the label. This method per se is widely practiced under
the name of EMIT.
Steric conjugates are used in steric hindrance methods for
homogeneous assay. These conjugates are synthesized by covalently
linking a low-molecular-weight hapten to a small analyte so that
antibody to hapten substantially is unable to bind the conjugate at
the same time as anti-analyte. Under this assay procedure the
analyte present in the test sample will bind anti-analyte, thereby
allowing anti-hapten to bind the conjugate, resulting in a change
in the character of the conjugate hapten, e.g., a change in
fluorescence when the hapten is a fluorophore.
Sandwich assays particularly are useful for the determination of
HRG2 or HRG2 antibodies. In sequential sandwich assays an
immobilized binding partner is used to adsorb test sample analyte,
the test sample is removed as by washing, the bound analyte is used
to adsorb labeled binding partner, and bound material is then
separated from residual tracer. The amount of bound tracer is
directly proportional to test sample analyte. In "simultaneous"
sandwich assays the test sample is not separated before adding the
labeled binding partner. A sequential sandwich assay using an
anti-HRG2 monoclonal antibody as one antibody and a polyclonal
anti-HRG2 antibody as the other is useful in testing samples for
HRG2 activity.
The foregoing are merely exemplary diagnostic assays for HRG2 and
antibodies. Other methods now or hereafter developed for the
determination of these analytes are included within the scope
hereof, including the bioassays described above.
The HRG2 polypeptides may be used for affinity purification of
receptors such as the p185.sup.HER2 and other similar receptors
that have a binding affinity for the HRG2, and more specifically
the HRG2-.alpha. and HRG2-.beta.. The HRG2-.alpha. and HRG2-.beta.,
may be used to form fusion polypeptides wherein the HRG2 portion is
useful for affinity binding to nucleic acids and to heparin.
The HRG2 polypeptides may be used as ligands for competitive
screening of potential agonists or antagonists for binding to the
p185.sup.HER2. Preferably, the HRG2-.alpha. or HRG2-.beta. is
detectibly labeled and a competition assay for bound p185.sup.HER2
is conducted using standard assay procedures.
The amino terminus of the cytoplasmic region of the HRG2-.alpha.
may be fused to the carboxy terminus of heterologous transmembrane
domains and receptors, to form a fusion polypeptide useful for
intracellular signaling of a ligand binding to the heterologous
receptor.
The methods and procedures described herein with HRG2-.alpha. may
be applied similarly to HRG2-.beta. and to other novel HRG2 ligands
and to their variants. All references cited in this specification
are expressly incorporated by reference. The following examples are
offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1
Preparation of Breast Cancer Cell Supernatants
The multiple types of HRG2 were isolated from the supernatant of
the human breast carcinoma MDA-MB-231. The HRG2 was released into
and isolated from the cell culture medium.
a) Cell Culture
MDA-MB-231, human breast carcinoma cells, obtainable from the
American Type Culture Collection (ATCC HTB 26), were initially
scaled-up from T25 cm.sup.2 tissue culture flasks to 890 cm.sup.2
plastic roller bottles (Corning, Corning, N.Y.) by serial passaging
and the seed train was maintained at the roller bottle scale. To
passage the cells and maintain the seed train, flasks and roller
bottles were first rinsed with phosphate buffered saline (PBS) and
then incubated with trypsin/EDTA (Sigma, St. Louis, Mo.) for 1-3
minutes at 37.degree. C. The detached cells were then pipetted
several times in fresh culture medium containing fetal bovine serum
(FBS), (Gibco, Grand Island, N.Y.) to break up cell clumps and to
inactivate the trypsin. The cells were finally split at a ratio of
1:10 into fresh medium, transferred into new flasks or bottles,
incubated at 37.degree. C., and allowed to grow until nearly
confluent. The growth medium in which the cells were maintained was
a combined DME/Ham's-F-12 medium formulation modified with respect
to the concentrations of some amino acids, vitamins, sugars, and
salts, and supplemented with 5% FBS. The same basal medium is used
for the serum-free ligand production and is supplemented with 0.5%
Primatone RL (Sheffield, Norwich, N.Y.).
b) Large Scale Production
Large scale MDA-MB-231 cell growth was obtained by using Percell
Biolytica microcarriers (Hyclone Laboratories, Logan, Utah) made of
weighted cross-linked gelatin. The microcarriers were first
hydrated, autoclaved, and rinsed according to the manufacturer's
recommendations. Cells from 10 roller bottles were trypsinized and
added into an inoculation spinner vessel which contained three
liters of growth medium and 10-20 g of hydrated microcarriers. The
cells were stirred gently for about one hour and transferred into a
ten-liter instrumented fermenter containing seven liters of growth
medium. The culture was agitated at 65-75 rpm to maintain the
microcarriers in suspension. The fermenter was controlled at
37.degree. C. and the pH was maintained at 7.0-7.2 by the addition
of sodium carbonate and CO.sub.02. Air and oxygen gases were
sparged to maintain the culture at about 40% of air saturation. The
cell population was monitored microscopically with a fluorescent
vital stain (fluorescein diacetate) and compared to trypan blue
staining to assess the relative cell viability and the degree of
microcarrier invasion by the cells. Changes in cell-microcarrier
aggregate size were monitored by microscopic photography.
Once the microcarriers appeared 90-100% confluent, the culture was
washed with serum-free medium to remove the serum. This was
accomplished by stopping the agitation and other controls to allow
the carriers to settle to the bottom of the vessel. Approximately
nine liters of the culture supernatant were pumped out of the
vessel and replaced with an equal volume of serum-free medium (the
same basal medium described as above supplemented either with or
without Primatone RL). The microcarriers were briefly resuspended
and the process was repeated until a 1000 fold removal of FBS was
acheived. The cells were then incubated in the serum-free medium
for 3-5 days. The glucose concentration in the culture was
monitored daily and supplemented with additions of glucose as
needed to maintain the concentration in the fermenter at or above 1
g/L. At the time of harvest, the microcarriers were settled as
described above and the supernatant was aseptically removed and
stored at 2-8.degree. C. for purification. Fresh serum-free medium
was replaced into the fermenter, the microcarriers were
resuspended, and the culture was incubated and harvested as before.
This procedure could be repeated four times.
Example 2
Purification of Growth Factor Activity
Conditioned media (10-20 liters) from MDA-MB-231 cells was
clarified by centrifugation at 10,000 rpm in a Sorvall Centrifuge,
filtered through a 0.22 micron filter and then concentrated 10-50
fold with a Minitan Tangential Flow Unit (Millipore Corp.) with a
10 kDa cutoff polysulfone membrane at room temperature.
Alternatively, media was concentrated with a 2.5 L Amicon Stirred
Cell at 4.degree. C. with a YM3 membrane. After concentration, the
media was again centrifuged at 10,000 rpm and the supernatant
frozen in 35-50 ml aliquots at -80.degree. C.
Heparin Sepharose was purchased from Pharmacia (Piscataway, N.J.)
and was prepared according to the directions of the manufacturer.
Five milliliters of the resin was packed into a column and was
extensively washed (100 column volumes) and equilibrated with
phosphate buffered saline (PBS). The concentrated conditioned media
was thawed, filtered through a 0.22 micron filter to remove
particulate material and loaded onto the heparin-Sepharose column
at a flow rate of 1 ml/min. The normal load consisted of 30-50 mls
of 40-fold concentrated media. After loading, the column was washed
with PBS until the absorbance at 280 nm returned to baseline before
elution of protein was begun. The column was eluted at 1 ml/min
with successive salt steps of 0.3M, 0.6M, 0.9M and 2.0M NaCl
prepared in PBS. Each step was continued until the absorbance
returned to baseline, usually 6-10 column volumes. Fractions of 1
milliliter volume were collected. All of the fractions
corresponding to each wash or salt step were pooled and stored for
subsequent assay in the MDA-MB-453 cell assay.
The majority of the tyrosine phosphorylation stimulatory activity
was found in the 0.6M NaCl pool which was used for the next step of
purification. Active fractions from the heparin-Sepharose
chromatography were thawed, diluted three fold with deionized
(MilliQ) water to reduce the salt concentration and loaded onto a
polyaspartic acid column (PolyCAT A, 4.6.times.100 mm, PolyLC,
Columbia, Md.) equilibrated in 17 mM Na phosphate, pH 6.8. All
buffers for this purification step contained 30% ethanol to improve
the resolution of protein on this column. After loading, the column
was washed with equilibration buffer and was eluted with a linear
salt gradient from 0.3M to 0.6M NaCl in 17 mM Na phosphate, pH 6.8,
buffer. The column was loaded and developed at 1 ml/min and 1 ml
fractions were collected during the gradient elution. Fractions
were stored at 4.degree. C. Multiple heparin-Sepharose and PolyCat
columns were processed in order to obtain sufficient material for
the next purification step. A typical absorbance profile from a
PolyCat A column is shown in FIG. 1. Aliquots of 10-25 .mu.L were
taken from each fraction for assay and SDS gel analysis.
Tyrosine phosphorylation stimulatory activity was found throughout
the eluted fractions of the PolyCAT A column with a majority of the
activity found in the fractions corresponding to peak C of the
chromatogram (salt concentration of approximately 0.45M NaCl).
These fractions were pooled and adjusted to 0.1% trifluoracetic
acid (TFA) by addition of 0.1 volume of 1% TFA. Two volumes of
deionized water were added to dilute the ethanol and salt from the
previous step and the sample was subjected to further purification
on high pressure liquid chromatography (HPLC) utilizing a C4
reversed phase column (SynChropak RP-4, 4.6.times.100 mm)
equilibrated in a buffer consisting of 0.1% TFA in water with 15%
acetonitrile. The HPLC procedure was carried out at room
temperature with a flow rate of 1 ml/min. After loading of the
sample, the column was re-equilibrated in 0.1% TFA/15%
acetonitrile. A gradient of acetonitrile was established such that
over a 10 minute period of time the acetonitrile concentration
increased from 15 to 25% (1%/min). Subsequently, the column was
developed with a gradient from 25 to 40% acetonitrile over 60 min
time (0.25%/min). Fractions of 1 ml were collected, capped to
prevent evaporation, and stored at 4.degree. C. Aliquots of 10 to
50 .mu.L were taken, reduced to dryness under vacuum (SpeedVac),
and reconstituted with assay buffer (PBS with 0.1% bovine serum
albumin) for the tyrosine phosphorylation assay. Additionally,
aliquots of 10 to 50 .mu.L were taken and dried as above for
analysis by SDS gel electrophoresis. A typical HPLC profile is
shown in FIG. 2.
A major peak of activity was found in fraction 17 (FIG. 2B). By SDS
gel analysis, fraction 17 was found to contain a single major
protein species which comigrated with the 45,000 dalton molecular
weight standard (FIGS. 2C, 3). In other preparations, the presence
of the 45,000 dalton protein comigrated with the stimulation of
tyrosine phosphorylation activity in the MDA-MB-453 cell assay. The
chromatographic properties of the 45,000 dalton protein were
atypical; in contrast to many other proteins in the preparation,
the 45,000 dalton protein did not elute from the reversed phase
column within 2 or 3 fractions. Instead, it was eluted over 5-10
fractions. This is possibly due to extensive post-translational
modifications.
a. Protein Sequence Determination
Fractions containing the 45,000 dalton protein were dried under
vacuum for amino acid sequencing. Samples were redissolved in 70%
formic acid and loaded into an Applied Biosystems, Inc. Model 470A
vapor phase sequencer for N-terminal sequencing. No discernable
N-terminal sequence was obtaining, suggesting that the N-terminal
residue was blocked. Similar results were obtained when the protein
was first run on an SDS gel, transblotted to ProBlott membrane and
the 45,000 dalton band excised after localization by rapid staining
with Coomassie Brilliant Blue.
Internal amino acid sequence was obtained by subjecting fractions
containing the 45,000 dalton protein to partial digestion using
either cyanogen bromide, to cleave at methionine residues, Lysine-C
to cleave at the C-terminal side of lysine residues, or Asp-N to
cleave at the N-terminal side of aspartic acid residues. Samples
after digestion were sequenced directly or the peptides were first
resolved by HPLC chromatography on a Synchrom C4 column (4000A,
2.times.100 mm) equilibrated in 0.1% TFA and eluted with a
1-propanol gradient in 0.1% TFA. Peaks from the chromatographic run
were dried under vacuum before sequencing.
Upon sequencing of the peptide in the peak designated number 15
(FIG. 2 [lysine C-15]), several amino acids were found on each
cycle of the run. After careful analysis, it was clear that the
fraction contained the same basic peptide with several different
N-termini, giving rise to the multiple amino acids in each cycle.
After deconvolution, the following sequence was determined (Seq. ID
#3): ##STR1## (Residues in brackets were uncertain while an X
represents a cycle in which is was not possible to identify the
amino acid.) The initial yield was 8.5 pmoles. This sequence
comprising 24 amino acids did not correspond to any previously
known protein. Residue 1 was later found from the cDNA sequence to
be Cys and residue 9 was found to be correct. The unknown amino
acids at positions 15 and 22 were found to be Cys and Ser,
respectively.
Sequencing on samples after cyanogen bromide and Asp-N digestions,
but without separation by HPLC, were performed to corroborate the
cDNA sequence. The sequences obtained are given in Table I and
confirm the sequence for the 45,000 protein deduced from the cDNA
sequence. The N-terminal of the protein appears to be blocked with
an unknown blocking group. On one occasion, direct sequencing of
the 45,000 dalton band from a PVDF blot revealed this sequence with
a very small initial yield (0.2 pmole)(Seq. ID #4):
(Residues which could not be determined are represented by "X",
while tentative residues are in parentheses). This corresponds to a
sequence starting at the serine at position 46 near the present
N-terminal of the FIG. 4 cDNA sequence; this suggests that the N
terminus of the 45,000 protein is at or before this point in the
sequence.
Example 3
Cloning and Sequencing of Human Heregulin 2
The cDNA cloning of the p185.sup.HER2 ligand was accomplished as
follows. A portion of the lysine C-15 peptide amino acid sequence
was decoded in order to design a probe for cDNA's encoding the 45
kD HRG2-.alpha. ligand. The following 39 residue long eight fold
degenerate deoxyoligonucleotide corresponding to the amino acid
sequence(Seq. ID #5)NH2- . . . AEKEKTFXVNGGE was chemically
synthesized(Seq. ID #6):
3'GCTGAGAAGGAGAAGACCTTCTGT/CGTGAAT/CGGA/CGGCGAG5'. The unknown
amino acid residue designated by X in the amino acid sequence was
assigned as cysteine for design of the probe. This probe was
radioactively phosphorylated and employed to screen by low
stringency hybridization an oligo dT primed cDNA library
constructed from human MDA-MB-231 cell mRNA in .lambda.gt10 (Huyng
et al., 1984, In DNA Cloning, Vol 1: A Practical Approach (D.
Glover, ed) pp.49-78. IRL Press, Oxford). Two positive clones
designated .lambda.gt10her16 and .lambda.gt10her13 were identified.
DNA sequence analysis revealed that these two clones were
identical.
The 2010 basepair cDNA nucleotide sequence of .lambda.gt10her16
(FIG. 4) contains a single long open reading frame of 669 amino
acids beginning with alanine at nucleotide positions 3-5 and ending
with glutamine at nucleotide positions 2007-2009. No translation
initiating methionine nor stop codon is found in the translated
sequence. Nucleotide sequence homology with the probe is found
between and including bases 681-719. Homology between those amino
acids encoded by the probe and those flanking the probe with the
amino acid sequence determined for the lysine C-15 fragment verify
that the isolated clone encodes at least the lysine C-15 fragment
of the 45 kD protein.
Hydropathy analysis shows the existence of a strongly hydrophobic
amino acid region including residues 287-309 (FIG. 4) indicating
that this protein contains a transmembrane domain and thus is
anchored to the membrane of the cell.
The 669 amino acid sequence encoded by the 2010 bp cDNA sequence
contains potential sites for asparagine-linked glycosylation
(Winzler, R. in Hormonal Proteins and Peptides, (Li, C.H. ed ) pp
1-15 Academic Press, New York (1973 )) at positions asparagine 164,
170, 208 and 437. A potential O-glycosylation site (Marshall, R. D.
(1974 ) Biochem. Soc. Symp. 40:17-26) is presented in the region
including a cluster of serine and threonine residues at amino acid
positions 209-218. Three sites of potential glycosaminoglycan
addition (Goldstein, L. A., et al. (1989) Cell 56:1063-1072) are
positioned at the serine-glycine dipeptides occuring at amino acids
42-43, 64-65 and 151-152.
This amino acid sequence shares a number of features with the
epidermal growth factor (EGF) family of transmembrane bound growth
factors (Carpenter, G., and Cohen, S. (1979 ) Ann. Rev. Biochem.
48: 193-216;Massenque, J. (1990) J. Biol. Chem. 265: 21393-21396)
including 1) the existence of a proform of each growth factor from
which the mature form is proteolytically released (Gray, A., Dull,
T. J., and Ullrich, A. (1983) Nature 303, 722-725; Bell, G. I. et
al., (1986) Nuc. Acid Res., 14: 8427-8446; Derynck, R. et al.
(1984) Cell: 287-297); 2) the conservation of six cysteine residues
characteristically positioned over a span of approximately 40 amino
acids (the EGF-like structural motif) (Savage, R. C., et al. (1973)
J. Biol.
Chem. 248: 7669-7672); HRG2-.alpha. cysteines 226, 234, 240, 254,
256 and 265 ); and, 3) the existence of a transmembrane domain
occuring proximally on the carboxy-terminal side of the EGF
homologous region (FIG. 4 and 6).
Alignment of the amino acid sequences in the region of the EGF
motif and flanking transmembrane domain of several human EGF
related proteins (FIG. 6) shows that between the first and sixth
cysteine of the EGF motif HRG2 is most similar (50%) to the heparin
binding EGF-like growth factor (HB-EGF) (Higashiyama, S. et al.
(1991) Science 251: 936-939). In this same region HRG2 is
.about.35% homologous to amphiregulin (AR) (Plowman, G. D. et al.,
(1990) Mol. Cell. Biol. 10: 1969-1981), .about.32% homologous to
transforming growth factor .alpha. (TGF .alpha.) (8), 27%
homologous with EGF (Bell, G. I. et al., (1986) Nuc. Acid Res., 14:
8427-8446); and 39% homologous to the schwanoma-derived growth
factor (Kimura, H., et al., Nature, 348:257-260, 1990). Disulfide
linkages between cysteine residues in the EGF motif has been
determined for EGF (Savage, R. C. et al. (1973) J. Biol. Chem. 248:
7669-7672). These disulfides define the secondary structure of this
region and demarcate three loops. By numbering the cysteines
beginning with 1 on the amino-terminal end, loop 1 is delineated by
cysteines 1 and 3; loop 2 by cysteines 2 and 4; and loop 3 by
cysteines 5 and 6. Although the exact disulfide configuration in
the region for the other members of the family has not been
determined, the strict conservation of the six cysteines, as well
as several other residues i.e., glycine 238 and 262 and arginine at
position 264, indicate that they too most likely have the same
arrangement. HRG2-.alpha. and EGF both have 13 amino acids in loop
1. HB-EGF, amphregulin (AR) and TGF .alpha. have 12 amino acids in
loop 1. Each member has 10 residues in loop 2 except HRG-.alpha.
which has 13. All five members have 8 residues in the third
loop.
EGF, AR, HB-EGF and TGF-.alpha. are all newly synthesized as
membrane anchored proteins by virtue of their transmembrane
domains. The proproteins are subsequently processed to yield mature
active molecules. In the case of TGF-.alpha. there is evidence that
the membrane associated proforms of the molecules are also
biologically active (Brachmann, R., et al. (1989) Cell 56:
691-700), a trait that may also be the case for HRG2-.alpha. . EGF
is synthesized as a 1168 amino acid transmembrane bound proEGF that
is cleaved on the amino-terminal end between arginine 970 and
asparagine 971 and at the carboxy-terminal end between arginine
1023 and histidine 1024 (Carpenter, G., and Cohen, S. (1979) Ann.
Rev. Biochem. 48: 193-216) to yield the 53 amino acid mature EGF
molecule containing the three loop, 3 disulfide bond signature
structure. The 252 amino acid proAR is cleaved between aspartic
acid 100 and serine 101 and between lysine 184 and serine 185 to
yield an 84 amino acid form of mature AR and a 78 amino acid form
is generated by NH.sub.2 -terminal cleavage between glutamine 106
and valine 107 (Plowman, G. D. et al., (1990) Mol. Cell. Biol. 10:
1969-1981). HB-EGF is processed from its 208 amino acid primary
translation product to its proposed 84 amino acid form by cleavage
between arginine 73 and valine 74 and a second site approximately
84 amino acids away in the carboxy-terminal direction (Higashiyama,
S., et al., and Klagsburn, M. (1991) Science 251: 936-939). The 160
amino acid preproform of TGF .alpha. is processed to a mature 50
amino acid protein by cleavages between alanine 39 and valine 40 on
one side and downstream cleavage between alanine 89 and valine 90
(Derynck et al., (1984) Cell: 38: 287-297). For each of the above
described molecules COOH-terminal processing occurs in the area
bounded by the sixth cysteine of the EGF motif and the beginning of
the transmembrane domain. The COOH-terminal processing site of
mature HRG2-.alpha. has not been defined, however several sites
seem plausible candidates ie, lysine 272-valine 273, lysine
278-alanine 279, or lysine 285-arginine 286 (FIG. 4). The
NH2-terminal end of HRG2-.alpha. likewise has not been determined;
preliminary amino acid sequence analysis of the mature molecule
indicates that processing may occur between methionine 45 and
serine 46 or further on toward the NH2-terminus.
HRG2-.alpha. may exert its biological function by binding to its
receptor and triggering the transduction of a growth modulating
signal. This it may accomplish as a soluble molecule or perhaps as
its membrane anchored form such as is sometimes the case with TGF
.alpha. (Brachmann, R., et al., (1989) Cell 56: 691-700).
Conversely, or in addition to stimulating signal transduction,
HRG2-.alpha. may be internalized by a target cell where it may then
interact with the controlling regions of other regulatory genes and
thus directly deliver its message to the nucleus of the cell. The
possibility that HRG2-.alpha. mediates some of its effects by a
mechanism such as this is suggested by the fact that a potential
nuclear location signal (Roberts, Biochem-Biophys Acta (1989) 1008:
263-280) exists in the region around the three lysine residues at
positions 58-60 (FIG. 4).
The isolation of full-length cDNA of HRG2-.alpha. is accomplished
by employing the DNA sequence of FIG. 4 to select additional cDNA
sequences from the cDNA library constructed from human MDA-MB-321.
Full-length cDNA clones encoding HRG2-.alpha. are obtained by
identifying cDNAs encoding HRG2-.alpha. longer in both the 3' and
5' directions and then splicing together a composite of the
different cDNAs. Additional cDNA libraries are constructed as
required for this purpose. Following are three types of cDNA
libraries that may be constructed: 1) Oligo-dT primed where
predominately stretches of polyadenosine residues are primed, 2)
random primed using short synthetic deoxyoligonucleotides
non-specific for any particular region of the mRNA, and 3)
specifically primed using short synthetic deoxyoligonucleotides
specific for a desired region of the mRNA. Methods for the
isolation of such cDNA libraries was previously described.
Example 4
Detection of HRG2-.alpha. mRNA Expression by Northern Analyses
Northern blot analysis of MDA-MB-231 and SKBR3 cell mRNA under high
stringency conditions shows at least five hybridizing bands in
MDA-MB-231 mRNA where a 6.4Kb band predominates: other weaker bands
are at 9.4, 6.9, 2.8 and 1.8Kb (FIG. 5). Only a faintly hybridizing
band at about 5.9kb is seen in SKBR3 mRNA. The existence of these
multiple messages in MDA-MB-231 cells indicates either alternative
splicing of the gene, various processing of the genes' primary
transcript or the existance of a transcript of another homologous
message. One of these messages may encode a soluble
non-transmembrane bound form of HRG2-.alpha.. Such messages (FIG.
5) may be used to produce cDNA encoding soluble non-transmembrane
bound form of HRG2-.alpha..
Example 5
Cell Growth Stimulation by Heregulin 2-.alpha.
Several different breast cancer cell lines expressing the EGF
receptor or the p185.sup.HER2 receptor were tested for their
sensitivity to growth inhibition or stimulation by ligand
preparations. The cell lines tested were: SKBR-3 (ATCC HTB 30), a
cell line which over expresses p.sub.185 HER2; MDA-MB-468 (ATCC HTB
132), a line which over expresses the EGF receptor; and MCF-7 cells
(ATCC HTB 22) which have a moderate level of p185.sup.HER2
expression. These cells were maintained in culture and passaged
according to established cell culture techniques. The cells were
grown in a 1:1 mixture of DMEM and F-12 media with 10% fetal bovine
serum. For the assay, the stock cultures were treated with trypsin
to detach the cells from the culture dish, and dispensed at a level
of 20000 cells/ well in a ninety-six well microtiter plate. During
the course of the growth assay they were maintained in media with
1% fetal bovine serum. The test samples were sterilized by
filtration through 0.22 micron filters and they were added to
quadruplicate wells and the cells incubated for 3-5 days at
37.degree. C. At the end of the growth period, the media was
aspirated from each well and the cells treated with crystal violet
(Lewis, G. et al., Cancer Research, 347:5382-5385, 1987). The
amount of crystal violet absorbance which is proportional to the
number of cells in each well was measured on a Flow Plate Reader.
Values from replicate wells for each test sample were averaged.
Untreated wells on each dish served as controls.
Results were expressed as percent of growth relative to the control
cells.
The purified HRG2-.alpha. ligand was tested for activity in the
cell growth assay and the results are presented in FIG. 7. At a
concentration of approximately 1 nM ligand, both of the cell lines
expressing the p.sub.185.sup.HER2 receptor (SKBR-3 and MCF-7)
showed stimulation of growth relative to the controls while the
cell type (MDA-MB-468) expressing only the EGF receptor did not
show an appreciable response. These results were consistent to
those obtained from the autophosphorylation experiments with the
various cell lines. These results established that the HRG2-.alpha.
ligand is specific for the p185.sup.HER2 receptor and does not show
appreciable interaction with the EGF receptor at these
concentrations.
Example 6
Isolation, Sequencing and Cloning of Heregulin 2-.beta.
After the final step of purification, HPLC chromatography on the C4
reversed phase column, the major peak of tyrosine phosphorylation
was found in the early fractions of the column (fraction 17).
However, other fractions showed activity in the phosphorylation
assay as well. By SDS reducing gel analysis, fraction 40 (see FIG.
2) from the C4 column consisted of two proteins of apparent
molecular masses of 14,000 and 12,000 daltons. An aliquot of this
fraction was dried down, electrophoresed on a 4-20% SDS
polyacrylamide gel and the resultant gel was blotted to PVDF
membrane as described earlier. Bands corresponding to the 14,000
and 12,000 dalton proteins were excised and subjected to protein
sequencing. Both bands yielded sequence, indicating that the
N-termini were not blocked. Initial yields for the 14,000 dalton
protein was approximately 7 pmoles, while that for the 12 kDa
protein was approximately 1 pmole. The sequence from the 12 kDa
protein was contained in the 14 kDa protein indicating that the 12
kDa protein is a proteolytic fragment of the larger protein. The
N-terminal sequence of this protein is (Seq. ID #7):
This sequence was found to be unique.
The cDNA cloning of the p185.sup.HER2 ligand HRG2-.beta. was
accomplished as follows. A portion of the N-terminal sequence was
decoded in order to design a probe for cDNAs encoding the 14 kD
HRG2-.beta. ligand. The following two 47 and 41 residue long eight
fold degenerate deoxyoligonucleotides corresponding to the amino
acid sequence above was chemically synthesized The two probe
sequences were:
(Sense strand)
5' CGG CAG CCC AAG TAC CCC XGG AAG TCC GCC CCC XGG XGG AAC AAG CT
3'(Seq. ID #8)
3' CC TTG TTC GAX CTG GTG ATA CGG TAG TAG TTG AAG GGG GAC 5'(Seq.
ID #9)
(Anti-sense strand)
In the sense strand, the Xs in triplets 7,12, and 13 may be either
C or A. In the anti-sense strand, the X in the third complete
triplet may be C or G. The sense strand containing 47 nucleotides
begins with arginine #3 in the N-terminal amino sequence of the 14
kDa HRG2-.beta.. The anti-sense strand containing 41 nucleotides
overlaps the sense strand as indicated by ten nucleotides indicated
by bold type. This partially double-stranded probe was
radioactively phosphorylated using Klenow polymerase I to extend
each DNA strand in the 3' direction using all 4 deoxyribonucleotide
triphosphates where the dCTP carried .alpha..sup.32 P. The
radiolabeled probe was then heat denatured and used to hybridize
under low strigency an oligo dT primed cDNA library constructed
from human MDA-MB231 cell mRNA in .lambda.gt10 (Huyng et al., 1984,
In DNA Cloning, Vol 1: A Practical Approach (D. Glover, ed)
pp.49-78. IRL Press, Oxford). Twenty-three positive clones were
identified.
The isolation of full-length cDNA of HRG2-.beta. is accomplished by
employing the DNA sequence encoding HRG2-.beta. to select
additional cDNA sequences from the cDNA library constructed from
human MDA-MB-231 cells. Full-length cDNA clones encoding
HRG2-.alpha. are obtained by identifying cDNAs encoding HRG2-.beta.
longer in both the 3' and 5' directions and then splicing together
a composite of the different cDNAs. Additional cDNA libraries are
constructed as required for this purpose. Following are three types
of cDNA libraries that may be constructed: 1) Oligo-dT primed where
predominately stretches of polyadenosine residues are primed, 2)
random primed using short synthetic deoxyoligonucleotides
non-specific for any particular region of the mRNA, and 3)
specifically primed using short synthetic deoxyoligonucleotides
specific for a desired region of the mRNA. Methods for the
isolation of such cDNA libraries was previously described.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 17 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 6 bases (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:1: CNCAAT6 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 bases (B) TYPE: nucleic
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:2: AATAAA6 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:3: AlaAlaGluLysGluLysThrPheCysValAsnGlyGlyGluXaa 151015
PheMetValLysAspLeuXaaAsnPro 2024 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:4: XaaGluXaaLysGluGlyArgGlyLysGlyLysGlyLysLysLys 151015
GluXaaGlyXaaGlyLys 2021 (2) INFORMATION FOR SEQ ID NO:5: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:5: AlaGluLysGluLysThrPheXaaValAsnGlyGlyGlu 151013 (2)
INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 42 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCTGAGAAGGAGAAGACCTTCTGTCGTGAATCGGACGGCGAG42 (2) INFORMATION FOR
SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino
acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:7:
LeuXaaArgGlnProLysTyrProArgLysSerAlaProArgArg 151015
AsnLysLeuAspHisTyrAlaIleIleLysPheProLeuThr 202529 (2) INFORMATION
FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 bases
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CGGCAGCCCAAGTACCCCNGGAAGTCCGCCCCCNGGNGGAACAAGCT47 (2) INFORMATION
FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 bases
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCTTGTTCGANCTGGTGATACGGTAGTAGTTGAAGGGGGAC41 (2) INFORMATION FOR SEQ
ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2010 bases (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGGCGCGAGCGCCTCAGCGCGGCCGCTCGCTCTCCCCC38
AlaArgAlaProGlnArgGlyArgSerLeuSerPro 1510
TCGAGGGACAAACTTTTCCCAAACCCGATCCGAGCCCTT77
SerArgAspLysLeuPheProAsnProIleArgAlaLeu 152025
GGACCAAACTCGCCTGCGCCGAGAGCCGTCCGCGTAGAG116
GlyProAsnSerProAlaProArgAlaValArgValGlu 3035
CGCTCCGTCTCCGGCGAGATGTCCGAGCGCAAAGAAGGC155
ArgSerValSerGlyGluMetSerGluArgLysGluGly 404550
AGAGGCAAAGGGAAGGGCAAGAAGAAGGAGCGAGGCTCC194
ArgGlyLysGlyLysGlyLysLysLysGluArgGlySer 5560
GGCAAGAAGCCGGAGTCCGCGGCGGGCAGCCAGAGCCCA233
GlyLysLysProGluSerAlaAlaGlySerGlnSerPro 657075
GCCTTGCCTCCCCGATTGAAAGAGATGAAAAGCCAGGAA272
AlaLeuProProArgLeuLysGluMetLysSerGlnGlu 808590
TCGGCTGCAGGTTCCAAACTAGTCCTTCGGTGTGAAACC311
SerAlaAlaGlySerLysLeuValLeuArgCysGluThr 95100
AGTTCTGAATACTCCTCTCTCAGATTCAAGTGGTTCAAG350
SerSerGluTyrSerSerLeuArgPheLysTrpPheLys 105110115
AATGGGAATGAATTGAATCGAAAAAACAAACCACAAAAT389
AsnGlyAsnGluLeuAsnArgLysAsnLysProGlnAsn 120125
ATCAAGATACAAAAAAAGCCAGGGAAGTCAGAACTTCGC428
IleLysIleGlnLysLysProGlyLysSerGluLeuArg 130135140
ATTAACAAAGCATCACTGGCTGATTCTGGAGAGTATATG467
IleAsnLysAlaSerLeuAlaAspSerGlyGluTyrMet 145150155
TGCAAAGTGATCAGCAAATTAGGAAATGACAGTGCCTCT506
CysLysValIleSerLysLeuGlyAsnAspSerAlaSer 160165
GCCAATATCACCATCGTGGAATCAAACGAGATCATCACT545
AlaAsnIleThrIleValGluSerAsnGluIleIleThr 170175180
GGTATGCCAGCCTCAACTGAAGGAGCATATGTGTCTTCA584
GlyMetProAlaSerThrGluGlyAlaTyrValSerSer 185190
GAGTCTCCCATTAGAATATCAGTATCCACAGAAGGAGCA623
GluSerProIleArgIleSerValSerThrGluGlyAla 195200205
AATACTTCTTCATCTACATCTACATCCACCACTGGGACA662
AsnThrSerSerSerThrSerThrSerThrThrGlyThr 210215220
AGCCATCTTGTAAAATGTGCGGAGAAGGAGAAAACTTTC701
SerHisLeuValLysCysAlaGluLysGluLysThrPhe 225230
TGTGTGAATGGAGGGGAGTGCTTCATGGTGAAAGACCTT740
CysValAsnGlyGlyGluCysPheMetValLysAspLeu 235240245
TCAAACCCCTCGAGATACTTGTGCAAGTGCCAACCTGGA779
SerAsnProSerArgTyrLeuCysLysCysGlnProGly 250255
TTCACTGGAGCAAGATGTACTGAGAATGTGCCCATGAAA818
PheThrGlyAlaArgCysThrGluAsnValProMetLys 260265270
GTCCAAAACCAAGAAAAGGCGGAGGAGCTGTACCAGAAG857
ValGlnAsnGlnGluLysAlaGluGluLeuTyrGlnLys 275280285
AGAGTGCTGACCATAACCGGCATCTGCATCGCCCTCCTT896
ArgValLeuThrIleThrGlyIleCysIleAlaLeuLeu 290295
GTGGTCGGCATCATGTGTGTGGTGGCCTACTGCAAAACC935
ValValGlyIleMetCysValValAlaTyrCysLysThr 300305310
AAGAAACAGCGGAAAAAGCTGCATGACCGTCTTCGGCAG974
LysLysGlnArgLysLysLeuHisAspArgLeuArgGln 315320
AGCCTTCGGTCTGAACGAAACAATATGATGAACATTGCC1013
SerLeuArgSerGluArgAsnAsnMetMetAsnIleAla 325330335
AATGGGCCTCACCATCCTAACCCACCCCCCGAGAATGTC1052
AsnGlyProHisHisProAsnProProProGluAsnVal 340345350
CAGCTGGTGAATCAATACGTATCTAAAAACGTCATCTCC1091
GlnLeuValAsnGlnTyrValSerLysAsnValIleSer 355360
AGTGAGCATATTGTTGAGAGAGAAGCAGAGACATCCTTT1130
SerGluHisIleValGluArgGluAlaGluThrSerPhe 365370375
TCCACCAGTCACTATACTTCCACAGCCCATCACTCCACT1169
SerThrSerHisTyrThrSerThrAlaHisHisSerThr 380385
ACTGTCACCCAGACTCCTAGCCACAGCTGGAGCAACGGA1208
ThrValThrGlnThrProSerHisSerTrpSerAsnGly 390395400
CACACTGAAAGCATCCTTTCCGAAAGCCACTCTGTAATC1247
HisThrGluSerIleLeuSerGluSerHisSerValIle 405410415
GTGATGTCATCCGTAGAAAACAGTAGGCACAGCAGCCCA1286
ValMetSerSerValGluAsnSerArgHisSerSerPro 420425
ACTGGGGGCCCAAGAGGACGTCTTAATGGCACAGGAGGC1325
ThrGlyGlyProArgGlyArgLeuAsnGlyThrGlyGly 430435440
CCTCGTGAATGTAACAGCTTCCTCAGGCATGCCAGAGAA1364
ProArgGluCysAsnSerPheLeuArgHisAlaArgGlu 445450
ACCCCTGATTCCTACCGAGACTCTCCTCATAGTGAAAGG1403
ThrProAspSerTyrArgAspSerProHisSerGluArg 455460465
TATGTGTCAGCCATGACCACCCCGGCTCGTATGTCACCT1442
TyrValSerAlaMetThrThrProAlaArgMetSerPro 470475480
GTAGATTTCCACACGCCAAGCTCCCCCAAATCGCCCCCT1481
ValAspPheHisThrProSerSerProLysSerProPro 485490
TCGGAAATGTCTCCACCCGTGTCCAGCATGACGGTGTCC1520
SerGluMetSerProProValSerSerMetThrValSer 495500505
ATGCCTTCCATGGCGGTCAGCCCCTTCATGGAAGAAGAG1559
MetProSerMetAlaValSerProPheMetGluGluGlu 510515
AGACCTCTACTTCTCGTGACACCACCAAGGCTGCGGGAG1598
ArgProLeuLeuLeuValThrProProArgLeuArgGlu 520525530
AAGAAGTTTGACCATCACCCTCAGCAGTTCAGCTCCTTC1637
LysLysPheAspHisHisProGlnGlnPheSerSerPhe 535540545
CACCACAACCCCGCGCATGACAGTAACAGCCTCCCTGCT1676
HisHisAsnProAlaHisAspSerAsnSerLeuProAla 550555
AGCCCCTTGAGGATAGTGGAGGATGAGGAGTATGAAACG1715
SerProLeuArgIleValGluAspGluGluTyrGluThr 560565570
ACCCAAGAGTACGAGCCAGCCCAAGAGCCTGTTAAGAAA1754
ThrGlnGluTyrGluProAlaGlnGluProValLysLys 575580
CTCGCCAATAGCCGGCGGGCCAAAAGAACCAAGCCCAAT1793
LeuAlaAsnSerArgArgAlaLysArgThrLysProAsn 585590595
GGCCACATTGCTAACAGATTGGAAGTGGACAGCAACACA1832
GlyHisIleAlaAsnArgLeuGluValAspSerAsnThr 600605610
AGCTCCCAGAGCAGTAACTCAGAGAGTGAAACAGAAGAT1871
SerSerGlnSerSerAsnSerGluSerGluThrGluAsp 615620
GAAAGAGTAGGTGAAGATACGCCTTTCCTGGGCATACAG1910
GluArgValGlyGluAspThrProPheLeuGlyIleGln 625630635
AACCCCCTGGCAGCCAGTCTTGAGGCAACACCTGCCTTC1949
AsnProLeuAlaAlaSerLeuGluAlaThrProAlaPhe 640645
CGCCTGGCTGACAGCAGGACTAACCCAGCAGGCCGCTTC1988
ArgLeuAlaAspSerArgThrAsnProAlaGlyArgPhe 650655660
TCGACACAGGAAGAAATCCAGG2010 SerThrGlnGluGluIleGln 665669 (2)
INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 669 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AlaArgAlaProGlnArgGlyArgSerLeuSerProSerArgAsp 151015
LysLeuPheProAsnProIleArgAlaLeuGlyProAsnSerPro 202530
AlaProArgAlaValArgValGluArgSerValSerGlyGluMet 354045
SerGluArgLysGluGlyArgGlyLysGlyLysGlyLysLysLys 505560
GluArgGlySerGlyLysLysProGluSerAlaAlaGlySerGln 657075
SerProAlaLeuProProArgLeuLysGluMetLysSerGlnGlu 808590
SerAlaAlaGlySerLysLeuValLeuArgCysGluThrSerSer 95100105
GluTyrSerSerLeuArgPheLysTrpPheLysAsnGlyAsnGlu 110115120
LeuAsnArgLysAsnLysProGlnAsnIleLysIleGlnLysLys 125130135
ProGlyLysSerGluLeuArgIleAsnLysAlaSerLeuAlaAsp 140145150
SerGlyGluTyrMetCysLysValIleSerLysLeuGlyAsnAsp 155160165
SerAlaSerAlaAsnIleThrIleValGluSerAsnGluIleIle 170175180
ThrGlyMetProAlaSerThrGluGlyAlaTyrValSerSerGlu 185190195
SerProIleArgIleSerValSerThrGluGlyAlaAsnThrSer 200205210
SerSerThrSerThrSerThrThrGlyThrSerHisLeuValLys 215220225
CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCys 230235240
PheMetValLysAspLeuSerAsnProSerArgTyrLeuCysLys 245250255
CysGlnProGlyPheThrGlyAlaArgCysThrGluAsnValPro 260265270
MetLysValGlnAsnGlnGluLysAlaGluGluLeuTyrGlnLys 275280285
ArgValLeuThrIleThrGlyIleCysIleAlaLeuLeuValVal 290295300
GlyIleMetCysValValAlaTyrCysLysThrLysLysGlnArg 305310315
LysLysLeuHisAspArgLeuArgGlnSerLeuArgSerGluArg 320325330
AsnAsnMetMetAsnIleAlaAsnGlyProHisHisProAsnPro 335340345
ProProGluAsnValGlnLeuValAsnGlnTyrValSerLysAsn 350355360
ValIleSerSerGluHisIleValGluArgGluAlaGluThrSer 365370375
PheSerThrSerHisTyrThrSerThrAlaHisHisSerThrThr 380385390
ValThrGlnThrProSerHisSerTrpSerAsnGlyHisThrGlu 395400405
SerIleLeuSerGluSerHisSerValIleValMetSerSerVal 410415420
GluAsnSerArgHisSerSerProThrGlyGlyProArgGlyArg 425430435
LeuAsnGlyThrGlyGlyProArgGluCysAsnSerPheLeuArg 440445450
HisAlaArgGluThrProAspSerTyrArgAspSerProHisSer 455460465
GluArgTyrValSerAlaMetThrThrProAlaArgMetSerPro 470475480
ValAspPheHisThrProSerSerProLysSerProProSerGlu 485490495
MetSerProProValSerSerMetThrValSerMetProSerMet 500505510
AlaValSerProPheMetGluGluGluArgProLeuLeuLeuVal 515520525
ThrProProArgLeuArgGluLysLysPheAspHisHisProGln 530535540
GlnPheSerSerPheHisHisAsnProAlaHisAspSerAsnSer 545550555
LeuProAlaSerProLeuArgIleValGluAspGluGluTyrGlu 560565570
ThrThrGlnGluTyrGluProAlaGlnGluProValLysLysLeu 575580585
AlaAsnSerArgArgAlaLysArgThrLysProAsnGlyHisIle 590595600
AlaAsnArgLeuGluValAspSerAsnThrSerSerGlnSerSer 605610615
AsnSerGluSerGluThrGluAspGluArgValGlyGluAspThr 620625630
ProPheLeuGlyIleGlnAsnProLeuAlaAlaSerLeuGluAla 635640645
ThrProAlaPheArgLeuAlaAspSerArgThrAsnProAlaGly 650655660
ArgPheSerThrGlnGluGluIleGln 665669 (2) INFORMATION FOR SEQ ID
NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:12: SerHisLeuValLysCysAlaGluLysGluLysThrPheCysVal 151015
AsnGlyGlyGluCysPheMetValLysAspLeuSerAsnProSer 202530
ArgTyrLeuCysLysCysGlnProGlyPheThrGlyAlaArgCys 354045
ThrGluAsnValProMetLysValGlnAsnGlnGluLysAlaGlu 505560
GluLeuTyrGlnLysArgValLeuThrIleThrGlyIleCysIle 657075
AlaLeuLeuValValGlyIleMetCysValValAlaTyrCysLys 808590
ThrLysLysGlnArg 95 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 91 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
AsnSerAspSerGluCysProLeuSerHisAspGlyTyrCysLeu 151015
HisAspGlyValCysMetTyrIleGluAlaLeuAspLysTyrAla 202530
CysAsnCysValValGlyTyrIleGlyGluArgCysGlnTyrArg 354045
AspLeuLysTrpTrpGluLeuArgHisAlaGlyHisGlyGlnGln 505560
GlnLysValIleValValAlaValCysValValValLeuValMet 657075
LeuLeuLeuLeuSerLeuTrpGlyAlaHisTyrTyrArgThrGln 808590 Lys 91 (2)
INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 82 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AsnAspCysProAspSerHisThrGlnPheCysPheHisGlyThr 151015
CysArgPheLeuValGlnGluAspLysProAlaCysValCysHis 202530
SerGlyTyrValGlyAlaArgCysGluHisAlaAspLeuLeuAla 354045
ValValAlaAlaSerGlnLysLysGlnAlaIleThrAlaLeuVal 505560
ValValSerIleValAlaLeuAlaValLeuIleIleThrCysVal 657075
LeuIleHisCysCysGlnVal 8082 (2) INFORMATION FOR SEQ ID NO:15: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 87 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:15: LysLysLysAsnProCysAsnAlaGluPheGlnAsnPheCysIle 151015
HisGlyGluCysLysTyrIleGluHisLeuGluAlaValThrCys 202530
LysCysGlnGlnGluTyrPheGlyGluArgCysGlyGluLysSer 354045
MetLysThrHisSerMetIleAspSerSerLeuSerLysIleAla 505560
LeuAlaAlaIleAlaAlaPheMetSerAlaValIleLeuThrAla 657075
ValAlaValIleThrValGlnLeuArgArgGlnTyr 808587 (2) INFORMATION FOR SEQ
ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 87 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:16:
LysLysLysAsnProCysAlaAlaLysPheGlnAsnPheCysIle 151015
HisGlyGluCysArgTyrIleGluAsnLeuGluValValThrCys 202530
HisCysHisGlnAspTyrPheGlyGluArgCysGlyGluLysThr 354045
MetLysThrGlnLysLysAspAspSerAspLeuSerLysIleAla 505560
LeuAlaAlaIleIleValPheValSerAlaValSerValAlaAla 657075
IleGlyIleIleThrAlaValLeuLeuArgLysArg 808587 (2) INFORMATION FOR SEQ
ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 86 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:17:
LysLysArgAspProCysLeuArgLysTyrLysAspPheCysIle 151015
HisGlyGluCysLysTyrValLysGluLeuArgAlaProSerCys 202530
IleCysHisProGlyTyrHisGlyGluArgCysHisGlyLeuSer 354045
LeuProValGluAsnArgLeuTyrThrTyrAspHisThrThrIle 505560
LeuAlaValValAlaValValLeuSerSerValCysLeuLeuVal 657075
IleValGlyLeuLeuMetPheArgTyrHisArg 808586
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